Lipoxin compounds and their use in treating cell proliferative disorders

ABSTRACT

Compounds having the active site of natural lipoxins, but a longer tissue half-life are disclosed. In particular, 15-epi-lipoxins and their use in ameliorating undesired cell proliferation, which characterizes diseases such as cancer, are also disclosed.

RELATED APPLICATIONS

This application is a continuation of and claims the benefit of commonlyowned U.S. Ser. No. 09/309,423 filed on May 11, 1999, now U.S. Pat. No.6,316,648, which is a divisional application of commonly owned U.S. Ser.No. 08/712,610 filed on Sep. 13, 1996, now U.S. Pat. No. 6,048,897,which is a continuation-in-part application of commonly owned U.S. Ser.No. 08/453,125 filed on May 31, 1995, now U.S. Pat. No. 5,648,512, whichis a divisional application of commonly owned U.S. Ser. No. 08/260,030filed on Jun. 15, 1994, now U.S. Pat. No. 5,441,951, which is acontinuation-in-part of commonly owned U.S. Ser. No. 08/077,300 filed onJun. 15, 1993, now abandoned, all of which are incorporated herein byreference.

GOVERNMENT SUPPORT

The work leading to this invention was supported in part by one or moregrants from the U.S. Government. The U.S. Government therefore may havecertain rights in the invention.

BACKGROUND

Lipoxins are a group of biologically active mediators derived fromarachidonic acid through the action of lipoxygenase (LO) enzyme systems.(Serhan, C. N. and Samuelsson, B. (1984) Proc. Natl. Acad. Sci. USA81:5335). Formation in human cell types is initiated by 5-LO or 15-LO.(Serhan, C. N. (1991) J. Bioenerg. Biomembr. 23:105). Single-cell typesgenerate lipoxins at nanogram levels during human neutrophil-plateletand eosinophil transcellular biosynthesis of eicosanoids. (Serhan, C. N.and Sheppard, K.-A. (1990) J. Clin. Invest. 85:772). LXs are conjugatedtetraene-containing eicosanoids that modulate cellular events in severalorgan systems.

Lipoxin A₄ (LXA₄) and lipoxin B₄ (LXB₄) are the two major lipoxins. Eachenhances protein kinase C (PKC) activity in nuclei of erythroleukemiacells at 10 nM (Beckman, B. S. et al. (1992) Proc. Soc. Exp. Biol. Med.201:169). Each elicits prompt vasodilation at nM levels (Busija, D. W.et al. (1989) Am. J. Physiol. 256:H468; Katoh, T. et al. (1992) Am. J.Physiol. 263 (Renal Fluid Electrolyte Physiol. 32):F436). Thevasodilatory effects of lipoxins are well-documented. For example,administration of LXA₄ in micromolar amounts via inhalation blocksbronchoconstriction in asthmatic patients. (Christie, P. E. et al.(1992) Am. Rev. Respir. Dis. 145:1281).

In the 10⁻¹⁰ M range, LXA₄ also stimulates cell proliferation incombination with suboptimal concentrations of granulocyte-macrophagecolony stimulating factor (GM-CSF) to induce myeloid bone marrow colonyformation (Stenke, L. et al. (1991) Biochem. Biophys. Res. Commun.180:255). LXA₄ also stimulates human mononuclear cell colony formation(Popov, G. K. et al. (1989) Bull. Exp. Biol. Med. 107:93).

LXA₄ inhibits chemotaxis of polymorphonuclear leukocytes (Lee, T. H. etal. (1991) Biochem. Biophys. Res. Commun. 180:1416). An equimolarcombination of lipoxins has been found to modulate the polymorphonuclearneutrophil-mesangial cell interaction in glomerular inflammation.(Brady, H. R. et al (1990) Am. J. Physiol. 809). Activation of thepolymorphonuclear neutrophils (PMN) includes the release of mediators ofstructural and functional abnormalities associated with the early stagesof glomerular inflammation. (Wilson, C. B. and Dixon, F. J. (1986) In:The Kidney, edited by B. M. Brenner and F. C. Rector. Philadelphia, Pa.:Saunders, p. 800-891).

Lipoxins act as antagonists to leukotrienes (LT), which are mediators ofinflammation. LXA₄ modulates LTC₄-induced obstruction of airways inasthmatic patients. (Christie, P. E. et al. (1992) Am. Rev. Respir. Dis.145:1281). LXA₄ inhibits LTD₄- and LTB₄-mediated inflammation in animalin vivo models. (Badr, K. F. et al (1989) Proc. Natl. Acad. Sci.86:3438; Hedqvist, P. et al. (1989) Acta Physiol. Scand. 137:571). Priorexposure to LXA₄ (nM) blocks renal vasoconstrictor actions of LTD₄(Katoh, T. et al. (1992) Am. J.Physiol. 263 (Renal Fluid ElectrolytePhysiol. 32) F436). Leukotriene-induced inflammation occurs, forexample, in arthritis, asthma, various types of shock, hypertension,renal diseases, allergic reactions, and circulatory diseases includingmyocardial infarction.

Although lipoxins are potent small molecules that could be administeredin vivo to treat a number of diseases and conditions, these moleculesare short-lived in vivo. Compounds having the same bio-activities asnatural lipoxins, but a longer in vivo half-life would be valuablepharmaceuticals.

SUMMARY OF THE INVENTION

This invention features substantially purified 15-epi-lipoxin compounds.In one embodiment, the 15-epi-lipoxin compound is15R-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid and inanother embodiment, this acid has a 5S,6R, configuration (15-epi-LXA₄).In other embodiments, the 15-epi-lipoxin compound is15R-5,14,15-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid, andthis acid has a 5S,14R configuration (15-epi-LXB₄). In still otherembodiments, the 15-epi-lipoxin compound is 15-hydroxyeicosatetraenoicacid (15-HETE), and this acid has a 15R configuration.

This invention also features lipoxin analogs, which have an activeregion that is the same or similar to natural lipoxin, but a metabolictransformation region which is more resistant to in vivo catabolism. Theinstant disclosed lipoxin analogs therefore have the biological activityof natural lipoxins, but a longer metabolic half-life. Certain of theinstant disclosed lipoxin analogs may additionally have an increased invivo potency, higher binding affinity to lipoxin receptors or enhancedbio-activity as compared to natural lipoxins.

Like natural lipoxins, the instant disclosed small molecules are highlypotent and biocompatible (i.e. non-toxic). However, unlike naturallipoxins, lipoxins analogs inhibit, resist, or more slowly undergometabolism and therefore have a longer pharmacological activity.Further, the instant disclosed compounds are more lipophilic thannatural lipoxins and therefore are more readily taken up by biologicalmembranes.

In addition, the invention features methods of ameliorating an undesiredproliferation of certain cells based on contacting the cells with aneffective amount of a substantially purified 15-epi-lipoxin compound. Inpreferred embodiments, the cells are undergoing cancerous or tumorousgrowth. Also in preferred embodiments, the cells are selected from thegroup consisting of: an epithelial cell, a leukocyte, an endothelialcell, and/or a fibroblast. In certain preferred embodiments of theinvention, cells are contacted in vivo. In another embodiment, cells arecontacted ex vivo.

The invention also features methods for ameliorating a cellproliferative disorder in a subject by administering an effective amountof a substantially purified 15-epi-lipoxin compound.

In another aspect, the invention features pharmaceutical compositionshaving the substantially purified 15-epi-lipoxin compound of the presentinvention and a pharmaceutically acceptable carrier. In a preferredembodiment, the 15-epi-lipoxin compound is in an amount effective toprevent an undesired proliferation of cells in a subject. In anotherembodiment, the pharmaceutical composition includes an effective amountof acetylsalicylic acid (ASA).

The invention further relates to diagnostic and research uses of thelipoxin compounds. Additional features and advantages of the inventionwill become more apparent from the following detailed description andclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing prostaglandin endoperoxide synthase (PGHS)and lipoxygenase (LO) expression in human tumor cell line (A549 cells)alveolar type II epithelial cells. Cells were grown for 24 h at 37° C.in T-75 cm² flasks in the presence or absence of Interleukin-1_(β)(IL-1_(β)) (1 ng/ml). Extracted total RNA (1 μg) was taken for ReverseTranscription (RT) and PCR using specific oligonucleotides for PGHS-1and -2, 15-, 12- and 5-LO and glyceraldehyde-3-phosphate dehydrogenase(GAPDH). Radioactive bands were quantified directly by phosphorimageranalysis, normalized to the expression of GAPDH and expressed as foldincrease in mRNA levels after exposure to IL-1_(β). The inset of FIG. 1Ashows 15-LO mRNA expression in human lung tissue and peripheral bloodmonocytes (PBM) with ND meaning 15-LO mRNA expression not detected.

FIG. 1B is a graph showing a RP-HPLC profile of [³H]-labeledmono-hydroxyeicosatetraenoic acids (HETEs) from permeabilizedIL-1_(β)-treated A549 cells (1.5×10⁶ cells/ml) exposed to[³H]-arachidonic acid (20 μM) for 20 min at 37° C. Products wereextracted and chromatographed using a linear gradient ofmethanol:H₂O:acetic acid (65:35:0.01; v/v/v) and methanol:acetic acid(99.9:0.1, v/v) at a flow rate of 1.0 ml/min. Arrows denoteco-chromatography of synthetic standards.

FIG. 2A is a graph showing the generation of 15-HETE. A549 cells (6×10⁶cells/flask) were treated with IL-1_(β) (1 ng/ml) for 24 h, subjected tofreeze-thaw (two cycles), exposed for 20 min to either vehicle (0.1%vol/vol ethanol (EtOH)), acetylsalicylic acid (ASA), the cytochrome P450inhibitor (17-octadecaynoic acid (17-ODYA), 5 μM) or the 5-LO inhibitor(Rev-5901 isomer, 5 μM) and incubated with arachidonic acid (20 μM) for20 min at 37° C. In some experiments, cells were heat-denatured (100°C., 60 min) before incubation. Incubations were stopped with addition ofmethanol (2v), and products were extracted for reversed phase(RP)-high-pressure liquid chromatography (HPLC). Data are means±SEM fromfour to six separate flasks. *, P<0.05 and **, P<0.01 for treatmentsversus control are shown.

FIG. 2B is a graph showing the time course of 15-HETE formation fromendogenous sources. A549 cells (1.5×10⁶ cells per ml) were grown for 48h in the absence or presence of IL-1_(β) (1 ng/ml) and incubated (30 minat 37° C.) in 4 ml HBSS with or without A₂₃₁₈₇ (5 μM). 15-HETE levelswere determined by RIA. Results represent the mean±SEM of threedifferent experiments determined by duplicate. *, P<0.05 for treatmentsversus vehicle are shown.

FIG. 3 is a graph showing the relative chiralities of 15-HETE triggeredby ASA. A549 cells (10⁷ cells per flask) were exposed to IL-1_(β) (1ng/ml) for 24 h, treated with vehicle (0.1% vol/vol ethanol) (□) or ASA(▪) for 20 min and then incubated (30 min, 37° C.) in HBSS containingarachidonic acid (20 μM) and A₂₃₁₈₇ (5 μM). Products werechromatographed by RP-HPLC (as in FIG. 1B) and the region containing15-HETE was collected, extracted with chloroform and treated withdiazomethane. Chiral analysis was performed with a Bakerbond DNBPG (seeMethods for details). Results are representative of two separateexperiments showing similar results. The inset of FIG. 3 shows the ratiobetween A549-derived 15R and 15S-HETE in the absence or presence (filledbars) of ASA.

FIG. 4A is a graph showing a RP-HPLC chromatogram of products fromepithelial cell-polymorphonuclear neutrophils (PMN) costimulation.Confluent A549 cells were exposed to IL-1_(β) (1 ng/ml) for 24 h,treated with ASA (20 min) and arachidonic acid (20 μM, 60 s) and eachincubated with freshly isolated PMN (A549 cell:PMN cell ratio of 1:8)followed by stimulation with ionophore A₂₃₁₈₇ (5 μM) in 4 ml of Hank'sbalanced salt solution (HBSS) for 30 min at 37° C. Products wereextracted and taken to RP-HPLC as described in the Methods section ofExample 5. The chromatogram was plotted at 300 nm and is representativeof n=6 experiments.

FIG. 4B is a graph showing on-line ultra-violet (UV) spectra of productsfrom the epithelial cell-PMN costimulation described in FIG. 4A.Material eluting beneath peak B was identified as predominantly15-epi-LXB₄.

FIG. 4C is a graph showing on-line UV spectra of products from theepithelial cell-PMN constimulation described in FIG. 4A. Materialeluting beneath the illustrated peaks was identified as predominantly15-epi-LXA₄.

FIG. 5A is a graph showing ASA modulating the formation oftetraene-containing lipoxins (lipoxins plus 15-epi-lipoxins) duringepithelial cell-PMN costimulation. A549 cells were exposed to IL-1_(β)(1 ng/ml, 24 h) and treated (20 min, 37° C.) with either vehicle (0.1%vol/vol) or ASA, before the addition of arachidonic acid (20 μM, 1 min)and freshly isolated PMN (A549/PMN cell ratio of 1:5). Costimulationswere carried out as in FIG. 4A. Results represent the mean±SEM from 3-5separate donors. The inset of FIG. 5A shows the effect of cell ratio ongeneration of tetraene-containing lipoxins (lipoxins plus15-epi-lipoxins) during co-incubations of A549 cells with PMN in theabsence (◯) or presence (▪) of ASA.

FIG. 5B is a graph showing ASA modulating the formation ofpeptidoleukotrienes (LTC₄ plus LTD₄) during epithelial cell-PMNcostimulation according to the conditions outlined in FIG. 5A. The insetof FIG. 5B shows the effect of cell ratio on generation ofpeptidoleukotrienes (LTC₄ plus LTD₄) during co-incubations of A549 cellswith PMN in the absence (◯) or presence (▪) of ASA.

FIG. 6A is a graph showing the effect of Lipoxin A₄ (LXA₄), Lipoxin B₄(LXB₄), Dexamethasone (DEX) and vehicle alone treatment on A549 cellnumber over time. A549 cells in 96-well plates were treated with eithervehicle (0.15% EtOH) or equimolar concentrations (10⁻⁶ M) of LXA₄, LXB₄or DEX for up to 96 hours at 37° C. At the indicated intervals, cellswere harvested for the3,(4,5-dimethylthiazoyl-2-yl)2,5(diphenyl-tetrazolium bromide) MTTassay. Data are means±SEM of 3-7 experiments performed in quadruplicate.*, P<0.05 and **, P<0.005 for compounds versus vehicle are shown.

FIG. 6B is a graph showing the effect of LXA₄, LXB₄, and DEX treatmenton the percent inhibition of A549 cell proliferation at varying A549cell concentrations. A549 cells were exposed to LXA₄, LXB₄ or DEX at theindicated concentrations for 72 hours at 37° C. Results are means±SEM of5-8 experiments performed in quadruplicate. Results are expressed as thepercent inhibition of proliferation relative to vehicle. *, P<0.05, **,P<0.025 and ***, P<0.005 for compounds versus vehicle are shown.

FIG. 7A is a graph showing the effect of LXA₄, LXB₄ and DEX on A549 cellDNA synthesis, as indicated by 3H-thymidine incorporation, where A549cells were grown for 72 hours in the presence of LXA₄, LXB₄ and DEX atvarying concentrations ranging between 5 nM to 500 nM. Twenty-four hoursbefore the assay, methyl-[³H]thymidine (2 μCi/ml) was added to eachwell. Cells were subsequently washed four times with DPBS²⁺ (4° C.),lysed with 0.25 N Sodium Hydroxide (NaOH), and radioactivityincorporation was monitored. Values represent mean±SEM of 3 differentexperiments performed in quadruplicate. Results are expressed as thepercent of [³H]thymidine incorporation relative to vehicle alone. *,P<0.05 and **, P<0.005 for compounds versus vehicle are shown.

FIG. 7B is a graph showing the effect of LXA₄, LXB₄ and DEX on theinhibition of A549 cells, where A 549 cells were seeded in 12-wellculture plates in the presence of LXA₄, LXB₄ and DEX (1 μM) and cellcounts were obtained at 72 hours by enumerating the trypan-excludingcells. Values represent mean±SEM of 3 different experiments. Results areexpressed as the percent inhibition of proliferation relative to buffer.*, P<0.05 for compounds versus vehicle are shown.

FIG. 8 is a diagram showing the proposed biochemical pathway forgenerating 15-epi-lipoxins. ASA-acetylated PGHS-2 and/or P450 activitiescontribute to 15R-HETE. Epithelial 15R-HETE undergoes transcellularconversion by Leukocyte 5-LO to a 15-epi-5(6)-epoxytetraeneintermediate, which is common to both 15-epi-LXA₄ and 15-epi-LXB₄.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following phrases and terms are defined as follows:

A “lipoxin compound” shall mean a natural lipoxin compound (lipoxin A₄or lipoxin B₄) and/or a lipoxin analog.

A “lipoxin analog” shall mean a compound which has an “active region”that functions like the active region of a “natural lipoxin”, but whichhas a “metabolic transformation region” that differs from naturallipoxin. Lipoxin analogs include compounds which are structurallysimilar to a natural lipoxin, compounds which share the same receptorrecognition site, compounds which share the same or similar lipoxinmetabolic transformation region as lipoxin, and compounds which areart-recognized as being analogs of lipoxin. Lipoxin analogs includelipoxin analog metabolites. The compounds disclosed herein may containone or more centers of asymmetry. Where asymmetric carbon atoms arepresent, more than one stereoisomer is possible, and all possibleisomeric forms are intended to be included within the structuralrepresentations shown. Optically active (R) and (S) isomers may beresolved using conventional techniques known to the skilled artisan. Thepresent invention is intended to include the possible diastereisomers aswell as the racemic and optically resolved isomers.

A preferred lipoxin compound for use in the subject invention is a“15-epi-lipoxin compound”. As used herein, “15-epi-lipoxin compound” isa lipoxin compound in which the absolute configuration at the 15 Carbonis R.

The term “15-epi-lipoxin compound” is intended to encompass precursors.The term “precursor” is intended to refer to chemical intermediates thatcan be converted in vivo, ex vivo and/or in vitro to form the15-epi-lipoxin compounds of the invention. The term “precursor” alsocontemplates prodrugs which are converted in vivo to the 15-epi-lipoxincompounds of the invention (see, e.g., R. B. Silverman, 1992, “TheOrganic Chemistry of Drug Design and Drug Action”, Academic Press, Chp.8). Examples of such prodrugs include, but are not limited to esters ofhydroxyls and/or carboxyl groups and/or compounds which can behydrolyzed or otherwise converted in vivo or, ex vivo and/or in vitrointo the 15-epi-lipoxin compounds of the present invention.

The terms “corresponding lipoxin” and “natural lipoxin” refer to anaturally-occurring lipoxin or lipoxin metabolite. Where an analog hasactivity for a lipoxin-specific receptor, the corresponding or naturallipoxin is the normal ligand for that receptor. For example, where ananalog is a LXA₄ analog having specific activity for a LXA₄ specificreceptor on differentiated HL-60 cells, the corresponding lipoxin isLXA₄. Where an analog has activity as an antagonist to another compound(such as a leukotriene), which is antagonized by a naturally-occurringlipoxin, that natural lipoxin is the corresponding lipoxin.

The term “active region” shall mean the region of a natural lipoxin orlipoxin analog, which is associated with in vivo cellular interactions.The active region may bind the “recognition site” of a cellular lipoxinreceptor or a macromolecule or complex of macromolecules, including anenzyme and its cofactor. Preferred lipoxin A₄ analogs have an activeregion comprising C₅-C₁₅ of natural lipoxin A₄. Preferred lipoxin B₄analogs have an active region comprising C5-C14 of natural lipoxin B4.

The term “recognition site” or receptor is art-recognized and isintended to refer generally to a functional macromolecule or complex ofmacromolecules with which certain groups of cellular messengers, such ashormones, leukotrienes, and lipoxins, must first interact before thebiochemical and physiological responses to those messengers areinitiated. As used in this application, a receptor may be isolated, onan intact or permeabilized cell, or in tissue, including an organ. Areceptor may be from or in a living subject, or it may be cloned. Areceptor may normally exist or it may be induced by a disease state, byan injury, or by artificial means. A compound of this invention may bindreversibly, irreversibly, competitively, noncompetitively, oruncompetitively with respect to the natural substrate of a recognitionsite.

The term “metabolic transformation region” is intended to refergenerally to that portion of a lipoxin, a lipoxin metabolite, or lipoxinanalog including a lipoxin analog metabolite, upon which an enzyme or anenzyme and its cofactor attempts to perform one or more metabolictransformations which that enzyme or enzyme and cofactor normallytransform on lipoxins. The metabolic transformation region may or maynot be susceptible to the transformation. A nonlimiting example of ametabolic transformation region of a lipoxin is a portion of LXA₄ thatincludes the C-13,14 double bond or the C-15 hydroxyl group, or both.

The term “detectable label molecule” is meant to include fluorescent,phosphorescent, and radiolabeled molecules used to trace, track, oridentify the compound or receptor recognition site to which thedetectable label molecule is bound. The label molecule may be detectedby any of the several methods known in the art.

The term “labeled lipoxin analog” is further understood to encompasscompounds which are labeled with radioactive isotopes, such as but notlimited to tritium (³H), deuterium (²H), carbon (¹⁴C), or otherwiselabeled (e.g. fluorescently). The compounds of this invention may belabeled or derivatized, for example, for kinetic binding experiments,for further elucidating metabolic pathways and enzymatic mechanisms, orfor characterization by methods known in the art of analyticalchemistry.

The term “inhibits metabolism” means the blocking or reduction ofactivity of an enzyme which metabolizes a native lipoxin. The blockageor reduction may occur by covalent bonding, by irreversible binding, byreversible binding which has a practical effect of irreversible binding,or by any other means which prevents the enzyme from operating in itsusual manner on another lipoxin analog, including a lipoxin analogmetabolite, a lipoxin, or a lipoxin metabolite.

The term “resists metabolism” is meant to include failing to undergo oneor more of the metabolic degradative transformations by at least one ofthe enzymes which metabolize lipoxins. Two nonlimiting examples of LXA₄analog that resists metabolism are 1) a structure which can not beoxidized to the 15-oxo form, and 2) a structure which may be oxidized tothe 15-oxo form, but is not susceptible to enzymatic reduction to the13,14-dihydro form.

The term “more slowly undergoes metabolism” means having slower reactionkinetics, or requiring more time for the completion of the series ofmetabolic transformations by one or more of the enzymes which metabolizelipoxin. A nonlimiting example of a LXA₄ analog which more slowlyundergoes metabolism is a structure which has a higher transition stateenergy for C-15 dehydrogenation than does LXA₄ because the analog issterically hindered at the C-16.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs.

The term “halogen” is meant to include fluorine, chlorine, bromine andiodine, or fluoro, chloro, bromo, and iodo.

The term “pharmaceutically acceptable salt” is intended to includeart-recognized pharmaceutically acceptable salts. These non-toxic saltsare usually hydrolyzed under physiological conditions, and includeorganic and inorganic bases. Examples of salts include sodium,potassium, calcium, ammonium, copper, and aluminum as well as primary,secondary, and tertiary amines, basic ion exchange resins, purines,piperazine, and the like. The term is further intended to include estersof lower hydrocarbon groups, such as methyl, ethyl, and propyl.

The term “pharmaceutical composition” comprises one or more lipoxinanalogs as active ingredient(s), or a pharmaceutically acceptablesalt(s) thereof, and may also contain a pharmaceutically acceptablecarrier and optionally other therapeutic ingredients. The compositionsinclude compositions suitable for oral, rectal, ophthalmic, pulmonary,nasal, dermal, topical, parenteral (including subcutaneous,intramuscular and intravenous) or inhalation administration. The mostsuitable route in any particular case will depend on the nature andseverity of the conditions being treated and the nature of the activeingredient(s). The compositions may be presented in unit dosage form andprepared by any of the methods well-known in the art of pharmacy. Dosageregimes may be adjusted for the purpose to improving the therapeuticresponse. For example, several divided dosages may be administered dailyor the dose may be proportionally reduced over time. A person skilled inthe art normally may determine the effective dosage amount and theappropriate regime. A lipoxin analog pharmaceutic composition can alsorefer to a combination comprising lipoxins, lipoxin analogs, and/orlipoxin metabolites, including metabolites of lipoxin analogs. Anonlimiting example of a combination is a mixture comprising a lipoxinanalog x which inhibits one enzyme which metabolizes lipoxins and whichoptionally has specific activity with a lipoxin receptor recognitionsite, and a second lipoxin analog y which has specific activity with alipoxin receptor recognition site and which optionally inhibits orresists lipoxin metabolism. This combination results in a longer tissuehalf-life for at least y since x inhibits one of the enzymes whichmetabolize lipoxins. Thus, the lipoxin action mediated or antagonized byy is enhanced.

The term “substantially pure or purified” lipoxin compounds are definedas encompassing natural or synthetic compounds of lipoxins having lessthan about 20% (by dry weight) of other biological macromolecules, andpreferably having less than about 5% other biological macromolecules(but water, buffers, and other small molecules, especially moleculesshaving a molecular weight of less than 5000, can be present. The term“purified” as used herein preferably means at least 80% by dry weight,more preferably in the range of 95-99% by weight, and most preferably atleast 99.8% by weight, of biological macromolecules of the same typepresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 5000, can be present).The term “pure” as used herein preferably has the same numerical limitsas “purified” immediately above. “Isolated” and “purified” do notencompass either natural materials in their native state or naturalmaterials that have been separated into components (e.g., in anacrylamide gel) but not obtained either as pure (e.g. lackingcontaminating proteins, or chromatography reagents such as denaturingagents and polymers, e.g. acrylamide or agarose) substances orsolutions.

The term “subject” is intended to include living organisms susceptibleto conditions or diseases caused or contributed to by inflammation,inflammatory responses, vasoconstriction, myeloid suppression and/orundesired cell proliferation. Examples of subjects include humans, dogs,cats, cows, goats, and mice. The term subject is further intended toinclude transgenic species.

The term “cell proliferative disorder” includes disorders involving theundesired proliferation of a cell. Non-limiting examples of suchdisorders include tumors, (e.g., brain, lung (small cell and non-smallcell), ovary, prostate, breast or colon) or other carcinomas or sarcomas(e.g., leukemia, lymphoma).

The term “ameliorated” in intended to include treatment for, preventionof, limiting of and/or inhibition of undesired cell proliferation and/ora cell proliferative disorder.

Lipoxin Compounds

The instant invention is based on the surprising finding thatsubstantially pure 15-epi-lipoxin compounds ameliorate undesired cellproliferation in a subject. The 15-epi-lipoxin compounds of the presentinvention include 15R-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid, in a 5S,6R configuration (15-epi-LXA₄). The15-epi-lipoxin compounds of the present invention also include15R-5,14,15-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid, in a5S,14R configuration (15-epi-LXB₄). The 15-epi-lipoxins of the presentinvention further include 15-hydroxyeicosatetraenoic acid, particularlyin a 15R configuration.

The instant invention is based on the surprising finding that lipoxinsare rapidly metabolized in a unique fashion by certain cells in vivo.Although other LO-derived products (e.g. leukotrienes) are metabolizedby ω-oxidation followed by β-oxidation (Huwyler et al., (1992) Eur. J.Biochem. 206, 869-879), the instant invention is based on the unexpectedfinding that lipoxins are metabolized by a series of oxidation andreduction reactions acting on certain sites of the lipoxin molecule. Forexample, LXA₄ metabolism has been found to occur, at least in part, viaoxidation of the C-15 hydroxyl to generate 15-oxo-LXA₄, reduction of theC-13,14 double bond to yield 13,14-dihydro-15-oxo-LXA₄ and furtherreduction to yield 13,14-dihydro-LXA₄. In LXB₄ and its natural isomersthe analogous oxidation occurs at the C-5 hydroxyl and reduction occursat the C-6,7 double bond.

Thus, the instant invention features lipoxin analogs having lipoxinactivity, but which are chemically modified to prevent dehydrogenationand therefore subsequent degradation in vivo. In these analogs, the C-1to C-13 portion of the natural lipoxin may or may not be conserved.Variations of the C-1 to C-13 portion include different cis or transgeometry as well as substitutions. The disclosed compounds isrepresented below by a structural genus, which is further divided intosubgenuses. Subgenuses included in each of the following two R groups isdenoted by a Roman numeral on the left of the page.

The instant lipoxins comprising an “active region” and a “metabolictransformation region” as both terms are defined herein are generally ofthe following structure:

In one embodiment, the lipoxin analogs of this invention have thefollowing structural formula I:

wherein X is R₁, OR₁, or SR₁;

wherein R₁ is

(i) hydrogen;

(ii) alkyl of 1 to 8 carbons atoms, inclusive, which may be straightchain or branched;

(iii) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z_(i), Z_(iii), and Z_(v) are each independently selected fromthe group consisting of —NO₂, —CN, —C(═O)—R₁, —SO₃H, and hydrogen;wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, and hydroxyl;

(vii) detectable label molecule; or

(viii) straight or branched chain alkenyl of 2 to 8 carbon atoms,inclusive;

wherein Q₁ is (C═O), SO₂ or (CN);

wherein Q₃ is O, S or NH;

wherein one of R₂ and R₃ is hydrogen and the other is

(a) H;

(b) alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chainor branched;

(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched; and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched;

wherein R₄ is

(a) H;

(b) alkyl of 1 to 6 carbon atoms, inclusive, which may be straight chainor branched;

wherein Y₁ or Y₂ is —OH, methyl, or —SH and wherein the other is

(a) H

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3; and Z is cyano, nitro, or halogen;

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;or

(d) alkoxy of 1 to 4 carbon atoms, inclusive;

or Y₁ and Y₂ taken together are

(a) ═N; or

(b) ═O;

wherein R₅ is

(a) alkyl of 1 to 9 carbon atoms which may be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 4 and R_(i) is

(i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or

(iii) substituted phenyl

wherein Z_(i), Z_(iii), and Z_(v) are each independently selected fromthe group consisting of hydrogen, —NO₂, —CN, —C(═O)—R₁, methoxy, and—SO₃H; and

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, and hydroxyl;

(c) R_(a)Q_(a)R_(b)

wherein Q_(a)=—O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may bestraight chain or branched;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are selected independently from the groupconsisting of

(i) H;

(ii) CH_(a)Z_(b) where a+b=3, a=0to3, b=0+3, and wherein any Z isindependently selected from the group consisting of halogen;

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched; and

wherein R6 is

(a) H;

(b) alkyl from 1 to 4 carbon atoms, inclusive, straight chain orbranched;

(c) halogen; but excluding the C-1 position amides, C-1 positionalkanoates, and pharmaceutically acceptable C-1 position salts of (5S,6R, 15S)-trihydroxy-7E, 9E, 11Z, 13E-eicosatetraenoic acid (LXA₄); andexcluding C-5, C-6, and C-15 position alkanoates of LXA₄.

In one embodiment of this invention, the lipoxin analogs have thefollowing structure II:

wherein X is R₁, OR₁, or SR₁;

wherein R₁ is

(i) hydrogen;

(ii) alkyl of 1 to 8 carbons atoms, inclusive, which may be straightchain or branched;

(iii) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z_(i), Z_(iii), and Z_(v) are each independently selected fromthe group consisting of —NO₂, —CN, —C(═O)—R₁, hydrogen, and —SO₃H;

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, and hydroxyl;

(vii) detectable label molecule, such as but not limited to fluorescentlabels; or

(viii) alkenyl of 2 to 8 carbon atoms, inclusive, straight chain orbranched;

wherein Q₁ is (C═O), SO₂ or (C═N);

wherein Q₃ is 0, S or NH;

wherein one of R₂ and R₃ is hydrogen and the other is

(a) H;

(b) alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chainor branched;

(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may bestraight chain or branched;

wherein R₄ is

(a) H;

(b) alkyl of 1 to 6 carbon atoms, inclusive, which may be straight chainor branched;

wherein Y₁ or Y₂ is —OH, methyl, —H or —SH and wherein the other is

(a) H;

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3

Z is cyano, nitro, or halogen including F, Cl, Br, I;

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

(d) alkoxy of 1 to 4 carbon atoms, inclusive;

or Y₁ and Y₂ taken together are

(a) ═N; or

(b) ═O;

wherein R₅ is

(a) alkyl of 1 to 9 carbon atoms which may be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 4 and R_(i) is

(i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or

(iii) substituted phenyl

wherein Z_(i), Z_(iii), and Z_(v) are each independently selected fromthe group consisting of hydrogen, —NO₂, —CN, —C(═O)—R₁, methoxy, and—SO₃H;

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, and hydroxyl;

(c) —R_(a)Q_(a)R_(b)

wherein Q_(a)=—O— or —S—; and

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may bestraight chain or branched;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are selected independently from the groupconsisting of

(i) H; and

(ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3

wherein any Z is selected from the group consisting of halogen.

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched.

In one embodiment of this invention, the lipoxin analogs have thefollowing structure III:

wherein X is R₁, OR₁, or SR₁;

wherein R₁ is

(i) hydrogen;

(ii) alkyl of 1 to 8 carbons atoms, inclusive, which may be straightchain or branched;

(iii) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z_(i), Z_(iii), and Z_(v) are each independently selected fromthe group consisting of —NO₂, —CN, —C(═O)—R₁, hydrogen, and —SO₃H;

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, and hydroxyl;

(vii) detectable label molecule; or

(viii) alkenyl of 2 to 8 carbon atoms, inclusive, straight chain orbranched;

wherein Q₁ is (C═O), SO₂ or (C═N);

wherein Q₃ is O, S or NH;

wherein one of R₂ and R₃ is hydrogen and the other is

(a) H;

(b) alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chainor branched;

(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may bestraight chain or branched;

wherein R₄ is

(a) H; or

(b) alkyl of 1 to 6 carbon atoms, inclusive, which may be straight chainor branched;

wherein Y₁ or Y₂ is hydroxyl, methyl, hydrogen or thiol and

wherein the other is

(a) H;

(b) CH_(a)Z_(b)

where a+b =3, a=0 to 3, b=0 to 3

Z is cyano, nitro, or halogen [including F, Cl, Br, I];

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

(d) alkoxy of 1 to 4 carbon atoms, inclusive;

or Y₁ and Y₂ taken together are

(a) ═N; or

(b) ═O; and

wherein R₅ is

(a) alkyl of 1 to 9 carbon atoms which may be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 4 and R_(i) is

(i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl;

(iii) substituted phenyl

wherein Z_(i), Z_(iii), and Z_(v) are each independently selected fromthe group consisting of hydrogen, —NO₂, —CN, —C(═O)—R₁, methoxy, and—SO₃H;

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, and hydroxyl;

(c) —R_(a)Q_(a)R_(b)

wherein Q_(a)=—O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may bestraight chain or branched; or

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are selected independently from the groupconsisting of

(i) H;

(ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3

wherein any Z is selected from the group consisting of halogen.

In another embodiment of this invention, lipoxin analogs have thefollowing structural formula IV:

wherein X is R₁, OR₁, or SR₁;

wherein R₁ is

(i) hydrogen;

(ii) alkyl of 1 to 8 carbons atoms, inclusive, which may be straightchain or branched;

(iii) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z_(i), Z_(iii), and Z_(v) are each independently selected fromthe group consisting of —NO₂, —CN, —C(═O)—R₁, methoxy, hydrogen, and—SO₃H;

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, and hydroxyl;

(vii) detectable label molecule; or

(viii) alkenyl of 2 to 8 carbon atoms, inclusive, straight chain orbranched;

wherein Q₁ is (C═O), SO₂ or (CN);

wherein Q₃ is O, S or NH;

wherein one of R₂ and R₃ is hydrogen and the other is

(a) H;

(b) alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chainor branched;

(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may bestraight chain or branched;

wherein R₄ is

(a) H; or

(b) alkyl of 1 to 6 carbon atoms, inclusive, which may be straight chainor branched;

wherein Y₁ or Y₂ is —OH, methyl, or —SH and wherein the other is

(a) H;

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3,

Z is cyano, nitro, or halogen;

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;or

(d) alkoxy of 1 to 4 carbon atoms, inclusive;

or Y₁ and Y₂ taken together are

(a) ═N; or

(b) ═O;

wherein R₅ is

(a) alkyl of 1 to 9 carbon atoms which may be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 4 and R_(i) is

(i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or

(iii) substituted phenyl

wherein Z_(i)Z_(iii), and Z_(v) are each independently selected from thegroup consisting of hydrogen, —NO₂, —CN, —C(═O)—R₁, methoxy, and —SO₃H;

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, and hydroxyl;

(c) R_(a)Q_(a)R_(b)

wherein Q_(a)=—O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is alkyl of 0 to 8 carbon atoms, inclusive, which may bestraight chain or branched;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are selected independently from the groupconsisting of

(i) H; or

(ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3, and

wherein any Z is selected from the group consisting of halogen; or

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched; and

wherein R₆ is

(a) H;

(b) alkyl from 1 to 4 carbon atoms, inclusive, straight chain orbranched; or

(c) halogen.

In another embodiment of this invention, lipoxin analogs have thefollowing structural formula V:

wherein R₁ is

(i) hydrogen;

(ii) alkyl of 1 to 8 carbons atoms, inclusive, which may be straightchain or branched;

(iii) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(iv) aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z_(i), Z_(iii,), and Z_(v) are each independently selected fromthe group consisting of —NO₂, —CN, —C(═O)—R₁, hydrogen, and —SO₃H;

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, and hydroxyl;

(vii) detectable label molecule; or

(viii) alkenyl of 2 to 8 carbon atoms, inclusive, straight chain orbranched;

wherein n=1 to 10, inclusive;

wherein R₂, R_(3a), and R_(3b) are independently selected from

(a) H;

(b) alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chainor branched;

(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched; and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched;

wherein Y₁ or Y₂ is —OH, methyl, hydrogen, or —SH and wherein the otheris

(a) H;

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3, and Z is cyano, nitro, or halogen;

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

(d) alkoxy of 1 to 4 carbon atoms, inclusive, straight chain orbranched;

or Y₁ and Y₂ taken together are

(a) ═N; or

(b) ═O;

wherein Y₃ or Y₄ is —OH, methyl, hydrogen, or —SH and wherein the otheris

(a) H;

(b) CH_(a)Z_(b)

wherein a+b=3, a=0 to 3, b=0 to 3, and any Z is cyano, nitro, orhalogen;

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

(d) alkoxy of 1 to 4 carbon atoms, inclusive, straight chain orbranched;

or Y₃ and Y₄ taken together are

(a) ═N; or

(b) ═O;

wherein Y₅ or Y₆ is —OH, methyl, hydrogen, or —SH and

wherein the other is

(a) H;

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3 Z is cyano, nitro, or halogen;

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

(d) alkoxy of 1 to 4 carbon atoms, inclusive, straight chain orbranched;

or Y₅ and Y₆ taken together are

(a) ═N; or

(b) ═O;

wherein R₅ is

(a) alkyl of 1 to 9 carbon atoms which may be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 4 and R_(i) is

(i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or

(iii) substituted phenyl

wherein Z_(i), Z_(iii), and Z_(v) are each independently selected fromthe group consisting of hydrogen, —NO₂, —CN, —C(═O)—R₁, and —SO₃H;

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, methoxy, and hydroxyl;

(c) R_(a)Q_(a)R_(b)

wherein Q_(a)=—O— or —S—; and

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is either alkyl of 0 to 8 carbon atoms, inclusive, whichmay be straight chain or branched or substituted phenyl;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are selected independently from the groupconsisting of

(i) H; or

(ii) CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0+3, and

wherein any Z is selected from the group consisting of halogen; or

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched; but excluding the C-1position amides, C-1 position alkanoates, and pharmaceuticallyacceptable C-1 position salts of (5S, 14R, 15S)-trihydroxy-6E, 8Z, 10E,12E-eicosatetraenoic acid (LXB₄); C-5, C-6, and C-5 position alkanoatesof LXB₄.

In another embodiment of this invention, lipoxin analogs have thestructural formula VI:

wherein R_(a) is selected from the group

(a) H; or

(b) alkyl of 1 to 8 carbon atoms;

wherein R_(b) selected from the group consisting of:

In another preferred embodiment of this invention, lipoxin analogs havethe following structural formula VII:

wherein R_(a) is selected from the group

(a) H; or

(b) alkyl of 1 to 8 carbon atoms;

wherein R_(b) and R_(c) are independently selected from the group

(a) H;

(b) hydroxyl, or thiol;

(c) methyl or halomethyls including —CF₃ and —CH₂F;

(d) halogen;

(e) alkoxy of 1 to 3 carbon atoms, including methoxy;

wherein R_(d) and R_(e) are selected independently from the group

(a) H;

(b) hydroxyl, or thiol;

(c) methyl or halomethyl including —CF₃ and —CH₂F;

(d) halogen;

(e) alkoxy of 1 to 3 carbon atoms, inclusive, including methoxy; or

(f) alkyl or haloalkyl of 2 to 4 carbon atoms, inclusive, which may bestraight chain or branched; but excluding the C-1 position amides, C-1position alkanoates, and pharmaceutically acceptable C-1 position saltsof (5S, 6R, 15S)-trihydroxy-7E, 9E, 11Z, 13E-eicosatetraenoic acid(LXA₄); C-5, C-6, and C-15 position alkanoates of LXA₄.

In another preferred embodiment of this invention, the lipoxin analogshave the structural formula VIII:

wherein R_(a) is selected from the group

(a) H; or

(b) alkyl of 1 to 8 carbon atoms;

wherein R_(b) and R_(c) are independently selected from the group

(a) H;

(b) hydroxyl or thiol;

(c) halomethyl, including CF₃;

(d) halogen;

(e) alkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched;or

(f) alkoxy of 1 to 3 carbon atoms, inclusive;

wherein R_(d) and R_(e) are selected independently from the group

(a) H;

(b) hydroxyl, or thiol;

(c) methyl or halomethyl including —CF₃ and —CH₂F;

(d) halogen;

(e) alkoxy of 1 to 3 carbon atoms, inclusive, including methoxy; or

(f) alkyl or haloalkyl of 2 to 4 carbon atoms, inclusive, which may bestraight chain or branched.

In another preferred embodiment of this invention, the lipoxin analogshave the structural formula IX:

wherein R_(a) is selected from the group

(a) H; or

(b) alkyl of 1 to 8 carbon atoms;

wherein R_(b) and R_(c) are independently selected from the group

(a) H;

(b) hydroxyl or thiol;

(c) halomethyl, including CF₃ and CH₂F;

(d) halogen;

(e) alkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched;

(f) alkoxy of 1 to 3 carbon atoms, inclusive; and

wherein R₅ is

(a) alkyl of 1 to 9 carbon atoms which may be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 4 and R_(i) is

(i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or

(iii) substituted phenyl

wherein Z_(i), Z_(iii), and Z_(v) are each independently selected fromthe group consisting of hydrogen, —NO₂, —CN, —C(═O)—R₁, and —SO₃H;

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, methoxy, and hydroxyl;

(c) —R_(a)Q_(a)R_(b)

wherein Q_(a)=—O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is either alkyl of 0 to 8 carbon atoms, inclusive, whichmay be straight chain or branched or substituted phenyl;

(d) —C(R_(iii))(R_(iv))—R_(i)

wherein R_(iii) and R_(iv) are selected independently from the groupconsisting of

(i) H; or

(ii) CH_(a)Z_(b) where a+b=3, a =0 to 3, b 0+3

wherein any Z is selected from the group consisting of halogen; or

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched.

In another preferred embodiment, the compounds have the structuralformula X:

wherein R_(a) is selected from the group

(a) H; or

(b) alkyl of 1 to 8 carbon atoms, inclusive, straight chain or branched;and

wherein R_(b) and R_(c) are independently selected from the group

(a) H;

(b) hydroxyl or thiol;

(c) halomethyl, including, for example, CF₃;

(d) halogen;

(e) alkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched;

(f) alkoxy of 1 to 3 carbon atoms, inclusive, including methoxy.

In another preferred embodiment, the compounds have the structuralformula XI:

wherein R_(a) is

(i) hydrogen;

(ii) alkyl of 1 to 8 carbons atoms, inclusive, which may be straightchain or branched; or

(iii) detectable label molecule;

wherein n=1 to 10, inclusive;

wherein Y₂, R_(3a), and R_(3b) are independently selected from

(a) H;

(b) alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chainor branched;

(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straightchain or branched; or

(e) R_(a)Q₂R_(b)

wherein Q₂ is —O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched; and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched;

wherein Y₁ is —OH, methyl, or —SH;

wherein Y₂ is

(a) H;

(b) CH_(a)Z_(b)

where a+b=3, a=0 to 3, b=0 to 3

Z is halogen; or

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

wherein Y₃ and Y₅ are independently selected from the group consistingof:

(a) H;

(b) CH_(a)Z_(b)

wherein a+b =3, a=0 to 3, b=0 to 3 and any Z is cyano, nitro, orhalogen; or

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

wherein Y₄ and Y₆ are independently selected from the group consistingof:

(a) H;

(b) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

(c) alkoxy of 1 to 4 carbon atoms, inclusive, straight chain orbranched; or

(d) hydroxyl or thiol; and

wherein R₅ is

(a) alkyl of 1 to 9 carbon atoms which may be straight chain orbranched;

(b) —(CH₂)_(n)—R_(i)

wherein n=0 to 3 and R_(i) is

(i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl;

(iii) substituted phenyl

wherein Z_(ii) and Z_(iv) are each independently selected from the groupconsisting of halogen, methyl, hydrogen, methoxy, and hydroxyl;

(c) R_(a)Q_(a)R_(b)

wherein Q_(a)=—O— or —S—;

wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, which maybe straight chain or branched;

wherein R_(b) is

(d) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogenatoms, inclusive, straight chain or branched; or but excluding the C-1position amides, C-1 position alkanoates, and pharmaceuticallyacceptable C-1 position salts of (5S, 14R, 15S)-trihydroxy-6E, 8Z, 10E,12E-eicosateraenoic acid (LXB₄);

C-5, C-6, and C-5 position alkanoates (acetates) of LXB₄.

In the most preferred embodiment of this invention, the compounds ofthis invention have the following structural formulas:

where R′ is H or CH₃;

and where the substituents at C* are in the R configuration.

In other preferred embodiments of this invention, the compounds of thisinvention have the following structural formulas:

where the substituents at the C* are in the R configuration.

Method for Making Lipoxin Compounds

Preferred compounds can be made as specifically described in thefollowing Example 1. Other compounds of this invention can be made bycombined strategies for lipoxin (LX) and prostaglandin analog synthesisusing standard chemical methods such as selective hydrogenation,Pd(0)-Cu(I) coupling, Wittig-type coupling, Sharpless epoxidation, andasymmetric reductions following coupling of the major intermediatesdescribed below and in the literature to generate the stable LX analogsof this invention. (Webber, S. E. et al. (1988) Adv. Exp. Med. Biol.229:61; R_(a)dutchel, B. and Vorbrüggen, H. (1985) Adv. ProstaglandinThromboxane Leukotriene Res. 14:263; and Nicolaou, K. C. et al. (1991)Angew Chem. Int. Ed. Engl. 30:1100). Geometrical variations can beaccomplished e.g. as described in U.S. Pat. No. 4,576,758 and Nicolaou,K. C. (1989) J. Org. Chem. 54: 5527.

As shown below, LX analog compounds comprising subgenus 1 in Scheme Ican be prepared as three major fragments (A, B, and C), which can thenbe combined to form the total molecule.

Synthesis of the epoxy-alcohol for precursor fragment 2 can be generatedwith substitutents R₂, R₃ and R4 selected from hydrogen, phenyl, halogenor methyl. Each of these respective epoxy-alcohols may be transformedinto phenyl urethane derivatives as 3.

with PhNCO, pyrimidine and CH₂Cl₂ followed by Lewis acid catalysis bySN² opening to give a 1,2 cyclic carbonate that contains the vicinaldiol at C-6 in the (R) configuration required for binding and C-5 in the(S) configuration also established for bioactivity and binding at arecognition site. These alcohols are next protected to generate theprecursor A fragment as 4.

These A fragments can now be coupled to the fragment B intermediate, aphosphonium bromide 5 as in Webber, S. E. et al. (1988) Adv. Exp. Med.Biol. 229:61 in gram quantities to generate the combined A+B fragmentproducts 6.

Fragment C intermediates from Scheme I are generated in parallel topreparation of A-B couplings. In these C fragments, substitutions at Y₁and/or Y₂ are methyl, methoxy, hydrogen, cyano, nitro, or halogen; seespecific example 3. Thus, carrying 15-methyl and/or, for example,16-methyl or 16-phenoxy-derivatives permits these substituted-LXA₄analogs to be not susceptible to dehydrogenation.

Thus, the C fragments carrying the preferred resistance to enzymaticoxidation and/or dehydrogenation may be converted by protection of keysites, followed by bromination to give vinyl bromide products offragment C such as 6b that is coupled to 6 by using catalytic amounts ofP(Ph₃)₄ and CuI to generate the complete backbone structure of the LXA₄analogs of genus formula I. This scheme is further illustrated by thefollowing Examples.

The compounds of this invention within subgenus formulas II and III maybe made synthesized in a similar manner.

Compounds in genuses II and III are generated by first individuallypreparing substituted compounds of fragment A that are each coupled asin Scheme I to individually prepared fragment B to generate 7 or A₁+B₁fragments possessing individual substitutions at X, Q₁, R₂, R₃, and R₄asindicated.

The C₁ fragment 8 carrying the acetylenic group C-14,15 and the ω-C-20end substitutions will each be generated as shown above for structure 6prostaglandin analogs and converted to their corresponding vinyl bromideproducts as in (KCN JAC 1985, Webber) to yield brominated products ofeach individual substituted fragment C₁ or 8 species that are suitablefor coupling to 20 using catalytic amounts of P(Ph₃)₄ and CuI togenerate the combined products of the acetylenic-LXA₄ analog class. Eachof the final products may then be subject to gradient RP-HPLC usingrapid diode array detection (as in Serhan, C. N., Methods in Enzymology)for purification. The presence of the modification at C-15 thru C-20 ofLXA₄ can alter metabolism by dehydrogenases and oxidases by providingsteric hindrance, stable prostaglandin analogs carrying C-15 to ω-endsubstitutions have been prepared and are not metabolized bydehydrogenases (Radüchel, B. and Vorbrüggen, H. (1985) Adv.Prostaglandin Thromboxane Leukotriene Res. 14:263 and Vorbüggen, H. etal. In: Chemistry, Biochemistry, and Pharmacological Activity ofProstanoids (Roberts, S. M., Scheinmann, F. eds.). Oxford: PergamonPress).

The cyclo-LXA₄ compounds of this invention within genus formula IV maybe made in the following manner.

The parent compound of this class is also subject to a similar totalsynthesis strategy and is assigned three main fragments A, B, and C instructure 30. A precursor for fragment A may be prepared by routes usedin Nicolaou, K. C. (1989) J. Org. Chem. 54:5527 to prepare 10 in thesynthesis of 7-cis,11-trans-LXA₄ methyl ester.

Fragment B in 30 can be obtained via the precursor 11 orsaligenin-([O-hydroxybenzylalcohol) as generated in (Vorbrüggen et al.,p. 353). The benzyl alcohol 11 is reacted with 10 (1: 1) in the presenceof NaH in DMF to give 12. This key intermediate is silylated in BSTFAfollowed by coupling with individual fragments designed for Cprecursors.

13 can then be coupled to vinyl brominate fragment C of given individualdesign by treating the bromite precursor with 4.0 equivalents of AgNO₃,then 7.0 equiv. of KCN, EtOH/THF/H₂O (1:1:1), 0→25° C., 2-4 h. theindividual products are then subject to Lindlar cat. for selectivecatalytic mild hydrogenation in CH₂Cl₂ 2-3 h to give individualcompounds belonging to the genus IV. Each can be saponified in LiOH/THFto give corresponding free acids after isolation by RP-HPLC.

The invention of genus IV compounds is illustrated further below, thesynthesis of 15(±)methyl-cyclo-LXA₄ methyl ester and corresponding freeacid.

The compounds of this invention within genus formula V may be made inthe following manner. Several systems studied with LXB₄ indicate thatseveral sites within the natural compound are required for bioactivity(Serhan, C. N. (1991) J. Bioenerg. and Biomembr. 23:105). These sitesinclude the C-14 alcohol in the (R) configuration and the double bond atC-8,9 of the tetraene in the cis configuration. In addition, based onmetabolic studies resulting in the instant invention, several keyaddition sites have been identified as being necessary to preserve LXB₄bioactivity. These include preserving the C-15 alcohol fromdehydrogenase activity (i.e., block 5-oxo-LXB₄ formation); maintainingboth the Δ8 bond and 14(R) alcohol; and preventing reduction of Δ6-7double bond and β/ω-oxidations of the resultant compounds.

Thus, genus V (14) maintains the regions of LXB₄ which are necessary forits bioactivity, but modifies the regions available to metabolicdegradation. Again, a retrosynthetic analysis gives priority to threekey fragments A, B and C designated in 14. Coupling of key intermediatesto generate members of the LXB₄ analog class uses standard techniques asoutlined for LXA₄ and its analogs namely, selective hydrogenation (togenerate 8-cis geometry); Pd(0)-Cn(I) coupling to join A & B fragmentscarrying unique substitutions; Wittig-type coupling to join C fragmentsthat carry the required substitutions and Sharpless epoxidation to yieldthe 14(R) vicinal alcohol (see ref. Nicolaou, K. C. et al. (1991) Angew.Chem. Int. Ed. Engl. 30:1100.), ref.; Weber, S. E. et al. (1988) Adv.Exp. Med. Biol. 229:61 Ed. Wong, P. K. and Serhan, C. N.) and thosecited within). Thus by using a similar strategy to LXA₄ analogs and theconstruction of native LXB₄ the specific analogs can be obtained.

The A fragments of 15 that carry substitutions at R₂, R₆, R₇ that can be(H, CH₃, OCH₃, phenyl, halo-substituted phenyl are generated by standardmethods from, for example 16 where ═CH₂ of increasing chain length.

Compound 16 is converted to the vinyl bromide 15 as in (Nicolaou K. C.et al. (1991) Angew. Chem. Int. Ed. Engl. 30: 1100-16.) via atrimethylsilyl acetylenic intermediate 17 that is reduced bypinanyl-9BBN then n-BuN₄NF in THF to yield 18 than is now submitted forbromination after protecting essential moieties such the alcohol to give15 (fragment A).

The fragment C in 14 is generated from compound 19 with the R₅substitutions as indicated.

The acetylenic alcohol 19 is then reduced in LAH followed by Sharplessasymmetric epoxidation to generate 20 that is isolated by RP-HPLC toyield the (+) isomer that is used to generate the required C-14 alcoholof LXB₄ analogs in the (R) configuration compound 20 is transformed tothe corresponding aldehyde 21 after protection of the substituted groupscarried at R₄ and R₅ as well as the alcohol using PCC in methylenechloride. (Nicolaou, K. C. et al. (1991) Angew. Chem. Int. Ed. Engl.30:1100).

The phosphonium salt 22 can be prepared as in Ref.; (Weber, S. E. et al.(1988) Adv. Exp. Med. Biol. 229:61, Ed. Wong, P. K. and Serhan, C. N.)and used here to generate the B-C fragment coupling via a Wittig-typecoupling to give 23 carrying the designated substitutions in the groupof 23 where R₄ and R₅ carry substitutions. This cis double bond of 23can be is isomerized to give the trans isomer using 12 as a catalyst togive the parent precursor form of 14 as 24.

Then coupling of 24 to 15 is accomplished by Pd(O)-Cu(I) coupling togive the acetylenic precursor of 14 designated 25. Following selectiveLindlar catalytic hydrogenation the individual LXB₄ analogs can befurther purified via RP-HPLC used the tetraene skeleton as a convenientmeans to isolate individual products employing rapid diode arraydetection (Serhan, C. N. (1990) Meth. Enzymol. 187:167).

Utilities

The compounds of this invention have the biological activity of naturalLXs, but are more resistant to degradation or alternatively inhibit thedegradation of natural LXs. The disclosed compounds therefore haveutility as pharmaceuticals for treating or preventing a number ofdiseases or conditions associated with inadequate or inappropriate LXmediated cellular response in a subject.

Based on the anti-proliferative effect of the disclosed 15-epi-lipoxincompounds, the invention provides methods for ameliorating undesiredcell proliferation by contacting cells with a pharmaceutical compositionincluding an effective amount of the substantially purified 15-epi-LXcompound and a pharmaceutically acceptable carrier. The cell can becontacted in vivo and/or in vitro. Alternatively, cells can be removedfrom a subject; contacted with the substantially purified 15-epi-lipoxincompound of the present invention ex vivo and implanted in the subject.The invention also provides a method for ameliorating a cellproliferative disorder in a subject including administering an effectiveamount of a substantially purified 15-epi-LX compound.

The effective amount is ordinarily the amount which is required toassure sufficient exposure to a target cell population. Such an amountwill ordinarily depend upon the nature of the 15-epi-LX compound, themode of administration, the severity of the undesired cell proliferationor cell proliferative disorder and other factors considered by a personof ordinary skill when determining a dosage regimen.

Target cells to be contacted can be undergoing cancerous and/or tumorousgrowth. Alternatively, target cells can be undergoing abnormal cellproliferation in response to a stimulus, such as restenosis; and/ortarget cells can consist of transformed cells having a genetic makeupaltered from that of the original cells.

Preferred target cells include epithelial cells, leukocytes, endothelialcells, and/or a fibroblasts.

Based on the stimulatory action of LXs on selected cells, the inventionalso provides methods for treating a subject with a myeloid suppressivedisorder by administering to the subject an effective amount of apharmaceutical composition comprising a LX analog. The effective amountis ordinarily that amount which is required to assure sufficientexposure to the target cell population. Such an amount will ordinarilydepend upon the nature of the analog, the mode of administration, theseverity of the myeloid suppression, and other factors considered by aperson of ordinary skill when determining a dosage regimen.

Therapeutic use of a cell proliferative LX analog also includes removingcells from a subject, stimulating cell growth in vitro, andreintroducing the enhanced cell preparation, in whole or in part, intothe subject. Additional therapeutic agents (e.g. cytokines such asGM-CSF) may be optionally used in conjunction with the LX duringstimulation or in conjunction with the introduction of the cellpreparation.

In another embodiment, the compounds of this invention are used to treator prevent inflammation or an inflammatory response. LXA₄ inhibits theactivation of leukocytes which are mediators of inflammation. TheLXA₄-induced effect includes inhibition of leukocyte migration,generation of reactive oxygen species, and the formation ofpro-inflammatory mediators involved in tissue swelling. (Raud, J. et al.(1991) Adv. Exp. Med. Biol. 314:185. Cell—Cell Interactions in theRelease of Inflammation Mediators vol. 314) LXB₄ exhibitsradioprotective actions, such as preventing diarrhea and ataxia, in anin vivo assay with mouse hematopoietic stem cells. (Walken, T. L.Jr.,(1988) J. Radiat. Res. 29:255)

The leukocyte-mediated inflammation or inflammatory responses cause orcontribute to a wide variety of diseases and conditions includingvarious forms of asthma and arthritis. Included within the presentinvention are inflammatory responses to physical injury, such asphysical trauma, radiation exposure, and otherwise.

In another embodiment, the compounds of this invention are used to treator prevent inflammation by antagonizing the action of leukotrienes. LXA₄inhibits LTB₄-induced inflammation, blocking both plasma leakage andleukocyte migration in an in vivo assay of the hamster cheek pouch.(Hedqvist, P. et al. (1989) Acta Physiol. Scand. 137: 571.) Plasmaleakage and leukocyte migration are key events in both wound healing andinflammation. LXA₄ also antagonizes LTD₄-induced renal hemodynamicactions and blocks the binding of LTD₄ to mesangial cells which areresponsible, in part, for regulating hemodynamics in the kidney. (Badr.K. F. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 438.) The compoundsof this invention may be administered to antagonize the action ofsulfidopeptide leukotrienes, such as LTD₄, LTC₄, and LTB₄.Leukotriene-mediated vasoconstrictive responses are associated withdiseases such as: asthma, anaphylactic reactions, allergic reactions,shock, inflammation, rheumatoid arthritis, gout, psoriasis, allergicrhinitis, adult respiratory distress syndrome, Crohn's disease,endotoxin shock, traumatic shock, hemmorrhagic shock, bowl ischemicshock, renal glomerular disease, benign prostatic hypertrophy,inflammatory bowl disease, myocardial ischemia, myocardial infarction,circulatory shock, brain injury, systemic lupus erythematosus, chronicrenal disease, cardiovascular disease, and hypertension.

In another embodiment, the compounds of this invention are used to treator prevent a vasocontractive response or condition. LXs induceendothelium-dependent vasodilation (LXA₄) (Lefer, A. M. et al (1988)Proc. Natl. Acad. Sci. USA 85:8340) and dilation of cerebral arteriolesin new born pigs in vivo (LXA₄ and LXB₄) (Busija, D. W. et al. (1989)Am. J. Physiol. 256:468.). Furthermore, LXA₄ induces rapid arteriolardilation in hamster cheek pouch in vivo (Dahlen, S.-E. et al. (1987)Acta Physiol. Scand. 130:643) and in the renal hemodynamics of the rat.(Badr, K. F. et al. (1987) Biochem. Biophys. Res. Commun. 145: 408).

Vasocontractive responses or conditions cause, contribute, or areassociated with diseases and conditions such as renal hemodynamicdiseases, including glomerular diseases, cardiovascular diseasesincluding hypertension, myocardial infarction, myocardial ischemia, andvascular diseases and gastrointestinal diseases.

Also encompassed by this invention is a method of screening LX analogsor other compounds to identify those having a longer tissue half-lifethan the corresponding natural LX. This method can be used to determinewhether the compound inhibits, resists, or more slowly undergoesmetabolism compared to the natural LX. This method is performed bypreparing at least one enzyme which metabolizes LXs, contacting thecompound with the enzyme preparation, and determining whether thecompound inhibits, resists, or more slowly undergoes metabolism by theenzyme. Cells having a LX recognition site, such as polymorphonuclearneutrophils, peripheral blood monocytes, and differentiated HL-60 cellsare among the appropriate sources for the enzyme preparation. The LXrecognition site may exist naturally, or be induced artificially, by adisease state, or by an injury. A non-limiting example ofartificially-induced LXA₄ recognition sites is the induction of suchsites in differentiated HL-60 cells.

In one embodiment, preparation of the enzymes comprised harvesting cellsand performing freeze-thaw lysis three times, followed byultracentrifugation to yield a 100,000 g supernatant. A cell-free100,000 g pellet may also be used. In addition, an enzyme preparationmay comprise any enzymes that do not participate in natural LXmetabolism, but perform transformations upon LXs similar or equivalentto those transformations performed by the enzyme or enzymes whichnaturally metabolize LXs. Nonlimiting examples of appropriate enzymesare 15-hydroxyprostaglandin dehydrogenase, cytochrome P-450 monogenasesfrom human leukocytes, and rat and human liver microsomes.

Characterization of LX metabolites included standard techniques such asextraction, chromatography, and quantitative HPLC followed by trimethylsilyl derivatization, O-methoxime derivatization and gaschromatography/mass spectroscopy analysis. The experimental details ofthis embodiment are described below in Example 1.

LX analogs can also be screened for binding activity with a LX receptorrecognition site, for example by contacting the compound with a receptorrecognition site and determining whether and to what degree the compoundbinds. Examples of kinetic binding assays include homologousdisplacement, competitive binding, isotherm, and equilibrium bindingassays.

The receptor recognition site may normally exist or it may be induced bya disease state, by an injury, or by artificial means. For example,retinoic acid, PMA, or DMSO may be used to induce differentiation inHL-60 cells. Differentiated HL-60 cells express LXA₄-specific receptorrecognition sites. Examples of other cells which may be screened for LXspecificity include PMN, epithelial cells, and peripheral bloodmonocytes.

Selection of competitive ligands will depend upon the nature of therecognition site, the structure of the natural substrate, any structuralor functional analogs of the natural substrate known in the art, andother factors considered by a skilled artisan in making such adetermination. Such ligands also include known receptor antagonists. Thecompounds of this invention may be radiolabelled with isotopes including²H, ³H, ¹³C, and ¹⁴C by standard techniques known in the art ofradiochemistry or synthetic chemistry.

In one embodiment of this method, the structural specificity of inducedLXA₄ recognition sites was assessed with LXB₄, LTC₄, LTB₄ andtrihydroxyheptanoic methyl ester. The experimental details of thisembodiment are described below in Example 2.

In addition, the compounds of this invention may be used to exertcertain actions on specific cell types as developmental models forinflammation and injury. For example, LXA₄ stimulates the mobilizationof intra-cellular Ca²⁺, lipid remodeling, and chemotaxis withoutaggregation in human PMN (Palmblad, J. et al. Biochem. Biophys. Res.Commun. (1987) 145: 168; Lee, T. H. et al. Clin.Sci. (1989) 77:195;Nigam, S. et al. J.Cell. Physiol. (1990) 143:512; Luscinskas, F. W. etal. (1990) Biochem. Pharmacol. 39:355). LXA₄ also blocks both LTB₄ andFMLP-induced responses, such as IP₃ generation. LXB₄ also stimulateslipid remodeling. LXA₄ activates isolated PKC, and is specific for theγ-subspecies of PKC which is found in the brain and spinal cord.(Hansson, A. et al. Biochem. Biophys. Res. Commun. (1986) 134: 1215;Shearman, M. S. et al. FEBS Lett. (1989) 245: 167); The publicationNicolaou, K. C. et al. Angew. Chem. Int. Ed. Engl. (1991) 30: 1100 andreferences cited within are expressly incorporated here by reference.

The present invention is further illustrated by the following exampleswhich should in no way be construed as being further limiting. Thecontents of all references and issued patents cited throughout allportions of this application including the background are expresslyincorporated by reference.

EXAMPLES Example 1 Synthesis of Lipoxin Analog Compounds

Preparation of the methyl ester precursor of compound 1:

To a solution of 3-methyl-3-trimethylsiloxy-1-bromo-1-octene (130 mg.0.44 mmol) in benzene (1.5 mL) was added n-propylamine (0.05 mL, 0.61mmol) and Pd(PPh₃)₄ (20 mg. 0.02 mmol) and the solution was protectedfrom light. It was then degassed by the freeze-thaw method and stirredat rt for 45 min. (7E, 9E, 5S, 6R) Methyl5,6-di(tert-butyldimethylsiloxy)-dodeca-7,9-diene-11-ynoate (183 mg.0.44 mmol) (compound 12) and copper iodide (14 mg. 0.07 mmol) were addedand the solution was one more time degassed by the freeze-thaw method.The mixture was stirred for 3 h at rt and quenched with saturatedaqueous solution of NH₄Cl and extracted with ether. It was then washedwith brine and dried over MgSO₄ and the solvent was evaporated. Flashcolumn chromatography (silica, 3% ether hexanes) afforded pure compoundas a colorless liquid (171 mg. 57% yield).

To a solution of the compound (171 mg. 0.25 mmol) in THF (0.5 mL) wasadded n-BuN₄F(0.9 mL. 0.90 mmol) and the mixture was stirred at rt. Thereaction was completed in 2 h at which time it was poured into water andextracted with ether. The ether extracts were washed with brine, driedover Na₂SO₄ and the solvent was evaporated. Flash column chromatography(silica 4% MeOH/CH₂Cl₂) afforded the methyl ester (24 mg.) together withsome of the corresponding lactone. HPLC retention time: 9:39 min(microsorb reverse phase, 4.6 mm×25 cm, C-18 column, MeOH/H₂O 70:30 flowrate 1 ml/min, UV detector at 300 nm). UV in MeOH: λ_(max)283, 294, 311nm. ¹H NMR (500 MHz CDCl₃) δ6.53 (dd. 15.2 10.9 Hz, 1H), 6.32 (dd,J=15.1, 11.0 Hz, 1H), 6.17 (d, J=15.9 Hz, 1H) 5.83 (dd. J=17.5, 2.1 Hz,1H), 5.80 (dd. J=15.2, 6.7 Hz, 1H), 5.72 (dd. J=17.0, 2.1 Hz, 1H), 4.14(m, 1H), 3.68-3.64 (m, 4H), 2.35-2.31 (m, 2H), 1.51-1.48 (m, 1H),1.43-1.42 (m, 2H), 1.30-1.23 (m, 15H) 0.85 (t, 3H). ¹³C NMR (126 MHz,CDCl₃) δ150.01, 140.18, 132.95, 132.26, 112.43, 107.50, 75.23, 73.76,42.49, 33.67, 32.17, 31.36, 27.96, 23.56, 22.58, 21.03, 14.03.

Preparation of the methyl ester precursor of compound 2:

A solution of the methyl ester precursor of compound 1 (3 mg. in CH₂Cl₂(1 ml) was mixed with Lindlar's catalyst (1 mg.) and placed under ahydrogen atmosphere. The mixture was stirred at rt in the dark followedby HPLC until about 80% conversion (1 h). Filtration over celiteevaporation of the solvent and separation by HPLC gave a pure methylester. HPLC retention time: 10:02 min (microsorb reverse phase. 10 mm×25cm C-18 column, MeOH/H₂O 70:30 flow rate 4 ml/min. UV detector at 300nm). UV in MeOH: η_(max) 287, 301, 315 nm.

Preparation of the methyl ester precursor of compound 3:

This compound was prepared similarly to the preparation of the methylester precursor of compound 1 (from3-cyclohexyl-3-trimethylsiloxy-1-bromo-1-octene). Desilylation of thiscompound was also performed in a similar manner to afford the methylester. HPLC retention time 8:02 min (microsorb reverse phase, 4.6 mm×25cm. C-18 column, MeOH/H₂O 70:30, flow rate 1 ml/min, UV detector at 300nm). UV in MeOH: λ_(max) 282, 293, 311 nm. ¹H NMR (360 MHz, CDCl₃) δ6.56(dd, 15.4, 10.9Hz, 1H), 6.33 (dd, J=15.2, 10.9 Hz, 1H), 6.13 (dd,J=15.8, 6.5 Hz, 1H), 5.81 (dd, J=15.2, 6.4 Hz, 1H), 5.80 (d, J=15.6 Hz,1H), 5.73 (dd, J=15.4, 2.1 Hz, 1H), 4.15 (br, 1H), 3.93-3.90 (m, 1H),3.67 (br, 1H), 3.65 (s, 3H), 2.34 (t, 2H), 1.82-1.65 (m, 10H), 1.46-1.38(m, 3H), 1.26-1.01 (m, 5H).

Preparation of the methyl ester precursor of compound 4:

Selective hydrogenation of the methyl ester precursor of compound 3,followed by HPLC purification gave the methyl ester precursor ofcompound 4. HPLC retention time: 9.72 min (microsorb reverse phase, 10mm×25 cm C-18 column, MeOH/H₂O 70:30 flow rate 4 ml./min. UV detector at300 nm), UV in MeOH: λ_(max) 288, 301, 315 nm. ¹H NMR (250 MHz, C₆D₆)δ6.66-6.89 (m, 2H), 5.95-6.24 (m, 4H), 5.55-5.66 (m, 2H), 3.82 (m, 1H),3.73 (m, 1H), 3.41 (m, 1H), 3.31 (s, 3H, OCH₃), 2.08 (t, 2 H, CH₂COO),1.00-1.81 (m, 18H).

The methylesters can be converted to corresponding alcohols usingstandard techniques.

Synthesis of 15(R)-15-methyl-LXA₄ and 15(±)methyl-LXA₄

Approximately 1 gm acetylenic ketone a is prepared using Friedel-Craftsacylation of bis(trimethylsilyl) acetylene with hexanoyl chloride and isreduced using (−)-pinayl-9-BBN to give the (S) alcohol in CH₃N₂ as inWebber, S. E. et al. (1988) Adv. Exp. Med. Biol. 229:61; Nicolaou, K. C.et al. (1991) Angew. Chem. Int. Ed. Engl. 30:1100; and Vorbrüggen, H. etal.: In: Chemistry, Biochemistry, and Pharmacological Activity ofProstanoids (Roberts, S. M., Scheinmann, F. eds.). Oxford: PergamonPress, to generate the methyl at C-15.

Alternatively, the keto group can be treated with CH₃MgBr (60→70° C.) asin Vorbrüggen, H. et al.: In: Chemistry, Biochemistry, andPharmacological Activity of Prostanoids (Roberts, S. M., Scheinmann, F.eds.). Oxford: Pergamon Press to yield the 15(±)methyl of b (2-5 g) indry CH₂Cl₂ (˜20 ml) at 0° C. with sequential additions of 2,6-lutidine(5.2 ml) and tert-butyldimethylsilyl triflate (6.9 ml). This reaction ismixed for 1 h and then diluted with 100 ml ether for aqueous extractionand drying with MgSO₄.

The product c is then coupled with d

that is generated as in Nicolaou, K. C. et al. (1991) Angew Chem. Int.Ed. Engl. 30:1100; Nicolaou, K. C. et al. (1989) J. Org. Chem. 54:5527and Webber, S. E. et al (1988) Adv. Exp. Med. Biol. 229:61. Structure dfrom fragment A in Scheme I is suspended in 4.0 equiv. of AgNO₃, then7.0 equiv. of KCN, containing EtOH:THF:H₂O (1:1:1), 0-25° C. for 2 h togenerate the C-methyl ester protected 15-methyl-LXA₄ analog that isconcentrated and saponified in THF with LiOH (2 drops, 0.1 M) at 4° C.12-24 h to give the corresponding free acid.

Synthesis of 16-dimethyl-LXA₄

This compound is generated using the similar strategy by coupling dabove with e vide supra, or f to generate the 15-phenyl-LXA₄ analog, org to generate the 17-m-chlorophenoxy-LXA₄ analogs.

The appropriate C fragments in Scheme I (i.e. e, f, g, h,) are eachprepared as reviewed in Radüchel, B. and Vorbrüggen, H. (1985) Adv.Prostaglandin Thromboxane Leukotriene Res. 14:263 for the knowncorresponding prostaglandin analogues. In h, R=H; Cl, methoxy orhalogen.

Synthesis of 13,14-acetylenic-LXA₄ and Halogen-containing Analogs

Using the A₂B₂ generated fragment from Scheme II, the corresponding C₂fragments are prepared for coupling. Structures i and k are generated asin Nicolaou, K. C. et al. (1989) J. Org. Chem. 54:5527 and methylated asin Radüchel, B. and Vorbrüggen, H. (1985) Adv. Prostaglandin ThromboxaneLeukotriene Res. 14:263 are coupled to 7 to yield these LX analogues.The materials may be subject to RP-HPLC for purification vide supra.

Synthesis of 14,15-acetylenic-LXA₄

The designated combined A₂B₂ fragment can be prepared from couplings offragments A₁ and B₁, illustrated in Route II to carry the structure of 7or 4 vide supra for coupling to fragment C₂. The precursor for the C₂fragment 1 can be prepared as in Radüchel, B. and Vorbrüggen, H. (1985)Adv. Prostaglandin Thromboxane Leukotriene Res. 14:263 for aprostaglandin analog.

Precursor m as prepared previously (Nicolaou, K. C. (1989) J. Org. Chem.54:5527) is added at 1.2 equiv. to 0.05 equiv. of Pd(PPh₃)₄, 0.16 equiv.of CuI, n-PrNH₂, in benzene with Me₂Al-carrying 1, 2-3 h RT to yield n.

The alcohol protecting groups TBDMS=R are removed with 10 equiv. ofHF-pyr, THF, 0-25° C. (4 h) followed by exposure to 3.0 equivalents ofEt₃N, MeOH, 25° C. 15 min to open acid-induced δ-lactones that usuallyform between C-1-carboxy and C-5 alcohol in the LXs (Serhan, C. N.(1990) Meth. Enzymol.187:167 and Nicolaou, K. C. (1989) J. Org. Chem.54:5527). After mild treatment with Lindlar cat. 5% by weight, theextracted material may be subjected to LiOH saponification in THF togenerate the free acid of the target molecule that can be subject tofurther purification by RP-HPLC gradient mobile phase as in (Serhan, C.N. et al. (1990) Meth. Enzymol. 187:167).

Synthesis of 15(±)methyl-cyclo-LXA₄

Compound o as the SiMe₃ derivative can be placed (˜1 gm) in a roundbottom 100 ml flask under an atmosphere enriched with argon in degassedbenzene (20 ml). To this add 3.0 equivalents of a vinyl bromide fragmentvide infra. This coupling reaction is carried out in catalytic amountsof Pd (PPh₃)₄ and CuI and can be monitored by injected aliquots of thissuspension into RP-HPLC monitored by UV abundance with a rapid scanningdiode. The progression line course 1-3 h at 23° C. after which thematerial is extracted with ethyl acetate: H₂O 4:1 v/v) and concentratedby rotoevaporation. The methyl ester can be saponified in LiOH/THF togive quantitative yields of the free carboxylic acid. Other derivativescan be prepared as above using fragment A with different fragment Bmoieties that have been substituted to give for example a dimethyl orother derivative. This can be obtained by taking the readily availableketone p and treating it with CH₃MgBr (60° C.) to generate q that canalso be coupled to fragment A as above using conventional techniquessuch as Pd(O)-Cu(I) coupling. Increased chain length from C-15 can alsobe obtained.

Synthesis of 5-Methyl-LXB₄ and 4,4-Dimethyl-LXB₄

The 5-methyl-LXB₄ hinders or retards 5-oxo-LXB₄ formation. Using thegeneral scheme outlined above, the A fragment can be constructed tocarry the 5-methyl in a vinyl bromide r precursor that is coupled to ajoined B+C fragment by Pd(0)-Cu(I) coupling.

The vinyl bromide r can be obtained from the s that contains eitherdimethyl or hydrogen substituents at its C-4 position. The protectedprecursor t containing fragments B+C is generated as reported inreference (Nicolaou K. C. et al. (1991) Angew. Chem. Int. Ed. Engl.30:1100-16.). Compound t is converted to s or 28 by coupling with theindicated vinyl bromide. Thus the target molecule can be generated byadding rat 1.0 equv. (≈1 gm) to a round bottom flask degassed containingEt₂NH as solvent with t injected in Et₂NH at 1.2 equiv. Pd(Ph₃P)₄ isadded at 0.02 equiv. to give the 8(9)-containing acetylenic precursormethyl ester of s.

The material is extracted and subject to rotoevaporation suspended inquinoline (0.5 eq) in CH₂Cl₂ and subject to hydrogenation using (10%;25° C.) Lindlar catalyst and a stream of H₂ gas to selectively reducethe acetylenic double bond at position 8. The formation of the tetraenecomponent of the methylester of 5-methyl-LXB₄ or 4-dimethyl-LXB₄ methylester can be monitored by RP-HPLC to assess completion of the reduction(i.e., 1-3 h). The methyl#esters are next saponified to theircorresponding free acids by treating the products with LiOH in THF 25 μlH₂O added at 0→24°, 8-24 h.

Example 2 Lipoxin A₄ Metabolism by Human Promyelocytic Leukemia Cellsand Monocytes: Half-life Assay

HL-60 cells were purchased from American Type Culture Collection(Rockville, Md.), and other cell culture reagents were from GIBCO (GrandIsland, N.Y.). Versene (EDTA) was from Whittaker Bioproducts(Walkersville, Md.). Synthetic 11,12-acetylenic LXA₄ methyl ester andLXs were from Cascade Biochemical (Reading, U.K.). 15(S)-15-m-PGE₁, PGE₁and 5-HETE were from Cayman Chemical Co. (Ann Arbor, Mich.).[11,12-³H]LXA₄ was prepared from 11,12-acetylenic LXA₄ using Lindlarcatalyst as a custom tritiation (NET-259, lot 0 2793-275, New EnglandNuclear, Boston, Mass.). Tritiated products were isolated using RP-HPLC(Fiore et al. (1992) J. Biol. Chem. 267:16168; Serhan, C. N. (1990)Meth. Enzymol. 187:167). Methoxyamine and AND were from Sigma ChemicalCompany (St. Louis, Mo.). Manganese dioxide and Adams reagent were fromAldrich Chemical Co. (Milwaukee, Wis.).

Human PMN were obtained from healthy volunteers by gradientcentrifugation of heparinized fresh venous blood (Böyum, A. (1986)Scand. J. Clin. Lab. Invest. 21:77). HL-60 cells were seeded in RPMIsupplemented with penicillin (100 U/ml), streptomycin (100 μ/ml), fetalbovine serum (10%) (Hyclone, Logan, Utah) and incubated (37° C. with 5%CO₂ atmosphere) in plastic 250 ml flasks. Individual flasks containing5×10⁻⁷ HL-60 cells/ml were incubated in the presence or absence ofphorbol 12-myristate 13-acetate (PMA) (10 or 16 nM, 24-27 h) andadherence was monitored for induction of macrophage-like phenotype as inCollins, S. J. (1987) Blood 70:1233. Peripheral blood monocytes wereobtained (Goldyne, M. E. et al. (1984) J. Biol. Chem. 259:8815 afterplating fresh mononuclear cells onto plastic petri dishes containing PBSwith glucose (1 mg/ml) for 1 h at 37° C. Non-adherent cells were removedand adherent mononuclear cells; were gently resuspended using Versene (7ml/plate) and washed in PBS. PMN (>98%), adherent monocytes (>95%) andHL-60 cells were enumerated by light microscopy, suspending in PBS forincubations, and <2-3% in each case were permeable to trypan blue. Forsome experiments, cell-free supernatants were prepared from HL-60 cellstreated with PMA (16 nM) for 24-72 h. After harvesting, thedifferentiated cells were washed, then subject to freeze-thaw lysis(repeated 3 times) and ultracentrifugation (100,000 g, 1 h).

Incubations with eicosanoids were stopped with cold methanol containingeither PGB₂ or 5-HETE as internal standards (5-HETE was used when15-oxo-ETE was quantified). Products were extracted using Sep-pak C18and routinely chromatographed as in Serhan, C. N. (1990) Meth. Enzymol.187:167. RP HPLC system consisted of an LKB gradient dual pump equippedwith an Altex Ultrasphere-ODS (4.6 mm×25 cm) column, flow rate 1 ml/mineluted (0-20 min) with methanol/H₂O/acetic acid (65:35:0.01) andmethanol/acetic acid (99.99/0.1) in a linear gradient (20-45 min) thatwas used to quantitate the ω-metabolites of LTB₄ (i.e. 20-COOH and20-OH-LTB₄) as well as LXA₄. Recovery of internal standards was 82.27.9, mean S.D. (n=13). Compounds I-IV were separated using an AltexUltrasphere-ODS column (10 mm×25 cm) eluted at a flow rate of 3.0 ml/minwith methanol/H₂O/acetic acid (60:40:0.01, v/v/v). Formation of15-oxo-ETE by 100,000 g supernatants (cf. Agins, A. P. et al. (1987)Agents Actions 21:397; Xun, C-Q. et al. (1991) Biochem. J. 279:553; andSok, D-E. et al. (1988) Biochem. Biophys. Res. Commun. 156:524) wasquantified after RP-HPLC using an ODS column (4.6 mm×25 cm) eluted withmethanol/H₂O/acetic acid (70:30:0.01, v/v/v) monitored at 280 nm with aflow rate of 1 ml/min. Monocyte-derived products were alsochromatographed using a Hypersil column (5 IL, 4 mm×300 mm) eluted withmethanol/H₂O/acetic acid (60.40:0.01, v/v/v) and a flow rate of 1ml/min. On-line spectra were recorded using a diode array detector(Hewlett-Packard 1040M series II) equipped with HPLC^(3D) ChemStationsoftware (DOS series). Spectra were acquired using step 4 nm, B_(w)=10nm, range=235-360 nm with a sampling interval of 1.28 sec.

GC/MS was performed with a Hewlett-Packard 5971A mass selective detectorquadrupole equipped with a HPG1030A workstation and GC 5890. The columnwas a HPUltra 2 (cross-inked 5% phenyl methyl silicone gum phase; 25m×0.2 mm×0.33 μm) and injections were made in the splitless mode inbis(TMS)trifluoroacetamide (BSTFA). The temperature program wasinitiated at 150° C. and reached 250° C. at 10 min and 325° at 20 min.Standard saturated fatty acid methyl esters C₁₆-C₂₆ gave the followingretention times (min:sec; mean of n=6). C₁₆, 8.03; C₁₈, 9.77; C₂₀,12.22; C₂₂, 16.11; C₂₄, 20.72; C₂₆, 23.62 that were used to calculaterespective C values of LX-derived metabolites as in Serhan, C. N. (1990)Meth. Enzymol. 187:167. Diazomethane was prepared and the methyl esterproducts were treated with BSTFA (Pierce Chemical Co., Rockford, Ill.)to obtain Me₃Si derivatives. Methyl ester O-methoxime derivatives wereprepared as in Kelly, R. W. and Abel, M. H. (1983) Biomed. MassSpectrom. 10:276. Catalytic hydrogenations were performed in methanol (1ml) with Adams reagent (Aldrich, Milwaukee, Wis.) by saturating theplatinum IV oxide (1-2 mg) with a stream of bubbling hydrogen (20 min,RT). After extraction, materials were treated with diazomethane followedby BSTFA (overnight; RT).

RESULTS

Metabolism of LXA₄: Intact neutrophils from peripheral blood of healthydonors did not significantly metabolize exogenous LXA₄ while cells fromthe same donors rapidly transformed LXA₄ via ω-oxidation. In contrast,PMA-treated HL-60 cells that displayed monocyte/macrophage-likecharacteristics rapidly transformed LXA₄. Within the first 60 s ofexposure, >70% of LXA₄ was metabolized. In the absence of PMA treatment,neither intact HL-60 cells (undifferentiated) nor their cell-freesupernatants (100,000×g) ft=orm LXA₄ (n=3).

Differentiated HL-60 cells incubated with LXA₄ converted this eicosanoidto several products. Labeled LXA₄ was transformed to four main productsthat carried tritium (denoted compounds I-IV), which were collected forfurther analysis.

Structures of compounds I-IV To obtain quantities of these compoundsenabling structural studies, their retention times in RP-HPLC wereestablished using the ³H-label elution profile to mark boundaries, andunlabeled samples pooled from several incubations were chromatographedand individually collected from within these regions for GC/MS analysis.Selected ion monitoring of the products obtained after treatment withdiazomethane and BSTFA revealed that compounds I-IV each displayedprominent ions at m/z 203 [—CH(OSiMe₃)—(CH₂)₃—COOCH₃] indicating thatcarbons 1 through 5 of LXA₄ (carboxylic carbon is number 1) were notmodified, although each product gave a different retention time thanLXA₄. The methyl ester, trimethylsilyl derivative of LXA₄ displayedprominent ions in its electron impact spectrum at m/z 203 (base peak)and 173, with its molecular ion at 582 (M⁺4). Other ions of diagnosticvalue in this derivative of LXA₄ are observed at m/z 171 (203-32), 409(M-173), 379 (M-203), 482 (M-100) and 492 (M-90) (Serhan, C. N. et al.(1984) Proc. Natl. Acad. Sci. USA 81:5335; and Serhan, C. N. (1990)Meth. Enzymol 187:167. It is noteworthy that the LXs in general areknown to give extremely weak molecular ion peaks (Serhan, C. N. et al.(1984) Proc. Natl. Acad. Sci. USA 81:5335). Nevertheless, compoundslabeled I & II also possess prominent ions at m/z 173(Me₃SiO⁺═CH—(CH₂)₄—CH₃) indicating that the carbon 15-20 fragment ofthese LXA₄-derived products was intact, while the ion at m/z 173 was notevident in compounds III and IV. Thus, the conclusion that compoundsI-IV are metabolites of LXA₄ was based upon: their physical properties(HPLC and GC/MS), the finding that they carry tritium label, as well asthe absence of these products in incubations with HL-60 cells nottreated with PMA.

Next, compounds III and IV were focused on since it appeared that theyrepresent metabolites with structural modifications in the carbon 15through 20 fragment of LXA₄. Since co-oxidation (hydroxylation at carbon20) was a possibility, ions that could result from the respective 20-OHand 20-COOH forms of LXA₄ after derivatization, namely m/z 261 and 217(Me₃SiO⁺═CH—(CH₂)₄—CH₂OSiMe₃ and Me₃SiO⁺═CH—(CH₂)₄—CO₂Me), were scannedin the acquired GC-MS data profiles. Neither III nor IV displayedprominent ions at either m/z 261 or 217 indicating that these productswere not likely the result of ω-oxidation.]

The mass spectrum (C value 24.3) of the Me₃Si derivative, methyl esterof compound III was obtained. Prominent ions in its spectrum wereobserved at m/z 203 (base peak, CH(OSiMe₃)—(CH₂)₃—COOCH₃), 171 (203-32;elimination of CH₃OH), 215 [(M-203)-90, elimination of trimethylsilanol(Me₃SiOH)] and 99 (O═C—(CH₂)₄—CH₃). Ions of lower intensity were a m/z508 (M⁺) and 418 (M-90; loss of Me₃SiOH). The presence of these ionssuggested that the material that coeluted with ³H-labeled compound IIIwas the 15-oxo-derivative of LXA₄. This is supported by several lines ofevidence, namely the virtual loss of the prominent ion at m/z 173(Me₃SiO⁺═CH—(CH₂)₄—CH₃), presence of m/z 99 (O═C—(CH₂)₄—CH₃), theabsence of a tetraene chromophore and appearance of a new chromophore atUV_(max) at 335-340 nm. The tetraenone chromophore was confirmed bytreating LXA₄ with MnO₂ in chloroform as used for prostaglandinconversion (Änggard, E. and Samuelsson, B. (1964) J. Biol. Chem.239:4097). Also, the mass spectrum of the catalytic hydrogenationproduct gave a C value of 25.1 with prominent ions at m/z 203 (basepeak), m/z 99 (66%), m/z 313 (M-203 or M—CH(OSiMe₃)—(CH₂)₃—COOCH₃; 35%)and m/z 171 (36%) with no prominent ion at m/z 173. Less intense ionswere at m/z 516 (M⁺) and m/z 426 (M-90). Thus, the upward shift of 8 amuand framentation of this saturated derivative were consistent with thegeneration of the corresponding 15-oxo-derivative.

To examine this LXA₄-derived product further, an aliquot of the materialeluting beneath the peak labeled III was treated with diazomethanefollowed by methoximation (as in Bergholte, J. M. et al. (1987) Arch.Biochem. Biophys. 257:444) and treatment with BSTFA. Its spectrum, Cvalue 25.4, showed prominent ions at m/z 203 (base peak), 171 (203-32;loss of CH₃OH) and 229 [M-128 or CH₃O—N═C—(CH₂)₄CH₃-(2×90)]. Ions oflower intensity were at ml/z 537 (M⁺), 466 (M-71, the αt-cleavage ionM—CH₂(CH₂)₃CH₃), 481 (M-56 or M—CH₂═CH—CH₂—CH₃, a McLaffertyrearrangement ion), 431 [M-106 (possibly loss of C₇H₅N⁺)], 401 [M-136(elimination of Me₃SiOH +CH₃+OCH₃) and 460 (M-77, loss of NOCH₃ plusMeOH). Again, an ion at m/z 173 that would have originated from analcohol-containing C-15 fragment (Me₃SiO⁺—CH—(CH₂)₄—CH₃) was virtuallyabsent in its spectrum. Thus, the ions present are consistent with themethyl ester, O-methoxime derivative generated from the15-oxo-containing derivative of LXA₄. Together the prominent ionsobserved with these different derivatives suggest that material elutingbeneath the peak labeled III was the 15-oxo-product of LXA₄ (i.e.15-oxo-LXA₄).

The mass spectrum of the methyl ester Me₃Si derivative (C value 26.0) ofcompound IV showed. prominent ions at m/z 203 (base peak,CH(OSiMe₃)—(CH₂)₃COOCH₃), 171 (203-32; loss of CH₃OH), 99(O═C—(CH₂)₄—CH₃) and 307 (M-203 or MCH(OSiMe₃)—(CH₂)₃—COOCH₃). Ions oflower intensity were at m/z 510 (M⁺), 420 (M-90, loss oftrimethylsilanol) and 208 (M-(99+203)). Its UV spectrum showed a tripletof absorbance with maxima at 259, 269 and 280 nm, consistent for aconjugated triene chromophore. The presence of these ions and UVspectrum suggest that IV was a dihydro-15-oxo-metabolite of LXA₄. Thisbasic structure was supported by the presence of the ion at m/z 99 thatis consistent with a keto group at position carbon 15, and the presenceof m/z 203 as base peak revealed that the alcohol groups at carbons 5and 6 remain intact. In addition, the absence of a trienone chromophore(λcal=310 nm) indicates that loss of a double bond was at Δ13-14position to give the observed triene chromophore. Together these resultsindicate that compound IV was 13,14-dihydro-15-oxo-LXA₄.

The methyl ester, Me₃SiO derivative of compound II (C value −25.4) gaveions at m/z 203 (base peak; CH(OSiMe₃)—(CH₂)₃COOCH₃), 173(Me₃SiO+═CH—(CH₂)₄—CH₃), 171 (203-32) and 584 (M⁺). Its molecular ionwas two mass units higher than the LXA₄ derivative. These ions and atriplet band of absorbance λ_(max)MeOH 259 nm, 269 and 282 nm suggestthat compound II was a dihydro-derivative of LXA₄. The methyl ester,Me₃SiO derivative of compound I from HL-60 cells gave two products inGC. The major one (C value=25.0) gave similar ions in its mass spectrumas LXA₄, but instead its molecular ion was at m/z 586 with ions alsopresent at m/z 555 (M-31) and 496 (M-90), indicating that two of thefour double bonds were reduced (not shown). However, identical productswere not observed with peripheral blood monocytes (vide infra), and thusthe HL-60 cell-derived materials from peak I were not furthercharacterized in the present experiments. The structures of I-IVindicate that LXA₄ is not metabolized by ω-oxidation by intactleukocytes but instead is both dehydrogenated at carbon 15 alcohol andalso transformed from a conjugated tetraene to triene structures. Takentogether these observations suggested that LXA₄ may be attacked byAND-dependent 15-prostaglandin dehydrogenase (5-PGDH), an enzyme knownto carry out similar reactions with prostanoids as substrate (Änggard,E. and Samuelsson, B. (1964) J. Biol. Chem. 239:4097, and reviewed inHansen, H. S. (1976) Prostaglandins 12:647).

15-PGDH activity was recently shown to be induced in HL-60 cells (Xun,C-Q. et al. (1991) Biochem. J. 279:553), and it apparently utilizes15-HETE as substrate with 92% efficiency compared to PGE₂ (Agins, A. P.et al. (1987) Agents Actions 21:397). Indeed, 100,000 g supernatantsprepared from PMA-treated HL-60 cells converted 15-HETE to 15-oxo-ETEindicating the presence of a dehydrogenase activity afterdifferentiation. LXA₄ competed for catalysis of 15-HETE giving aK_(i)=8.2±2.6 μM (S.E.M., n=6) calculated from Lineweaver-Burke plots.At equimolar concentrations of LXA₄ and 15-HETE, LXA₄ blocked 15-oxo-ETEformation by p50%. The relative conversion for LX compared to PGE₁ by100,000 g supernatants indicated that LXA₄, 11-trans-LXA₄ as well asLXB₄ but not 15-methyl-PGE₁ were converted. Together, these resultssuggest that LXA₄>11-trans-LXA₄>LXB₄ are substrates for 15-PGDH or anequivalent enzyme system.

Since PMA induces differentiation to monocyte-macrophage-like lineage ofHL-60 cells (Collins, S. J. (1987) Blood 70:1233), peripheral bloodmonocytes were incubated to determine if they metabolize LX. LXssdisplay potent actions with monocytes (Stenke, L. et al. (1991b)Biochem. Biophys. Res. Commun. 180:255) and these cells do not ω-oxidizeeicosanoids (Goldyne, M. E. et al. (1984) J. Biol. Chem. 259:8815). Whensuspensions of both intact monocytes (n=5) and permeabilized cells(freeze-thaw or saponin-treated, n=5) were exposed to LXA₄, it wasconverted to 15-oxo-LXA₄ and conjugated triene-containing products,13,14-dihydro-LXA₄ and 13,14-dihydro-15-oxo-LXA₄. As with differentiatedHL-60 cells, monocytes rapidly converted LXA₄ (>60%) within 30 s. Thetemporal relationships for formation of these metabolites in both intactand permeabilized monocytes were similar and suggest that 15-oxo-LXA₄metabolite is a transient intermediate. Also, in each monocytesuspension incubated with ³H-LXA₄ (d=33), 13,14-dihydro-15-oxo-LXA₄ and13,14-dihydro-LXA₄ were major products carrying radiolabel. It isnoteworthy that a product eluting before 13,14-dihydro-LXA₄ at 15.5-17min was observed that also displayed a triene chromophore and was likelythe 11-trans isomer of 13,14-dihydro-LXA₄ that results from cis-transisomerization encountered during work-up, The 1 l-cis double bond ofnative LXA₄ is labile and readily isomerizes to all-trans duringextraction and isolation (Romano, M. and Serhan, C. N. (1992)Biochemistry 31:8269).

Example 3 Binding Affinity to Lipoxin Receptors Analogs

Human promyelocytic leukemia cells (HL-60) were purchased from theAmerican Type Culture Collection (Rockville, Md.). RPMI medium and cellculture reagents were from GIBCO (Grand Island, N.Y.). Synthetic LXA₄,trihydroxyheptanoic acid (methyl ester), LXB₄, LTD₄, LTC₄ and LTB₄ werefrom Biomol (Plymouth Meeting, Pa.), and SKF 104353 was from Smith Klineand French Laboratories. ONO 4057 was from ONO Pharmaceutical Co., Ltd.(Osaka, Japan). [14,15-³H]LTB₄ (32.8 mCi/mmole), [1-¹⁴C]arachidonic acid(50.2 mCi/mmole), ³²PγATP (3,000 Ci/mmole), [9,10-³H(N)]palmitic acid(30.0 mCi/mmole), and [9,10-³H(N)]myristic acid (30.7 Ci/mmole) werepurchased from New England Nuclear (DuPont Co., Boston, Mass.).11,12-acetylenic LXA₄ was from Cascade Biochemicals (Oxford, UK).Microcentrifuge tube filters (0.45 μm cellulose acetate) were purchasedfrom PGC Scientific (Gaithersburg, Md.) and silicon oils were fromHarwick Chemical Corp. (Akron, Ohio) (d=1.05) and Thomas Scientific(Swedesboro, N.J.) (d=0.963), respectively. Nitroblue tetrazolium, PMA,DMSO, proteases, retinoic acid and Actinomycin D were purchased fromSigma (St. Louis, Mo.). Islet activating protein (IAP) was from LISTBiological Lab., Inc. (Campbell, Calif.). Plasticware, Whatman LK6D TLCplates and solvent (HPLC grade) were from Fisher (Springfield, N.J.).

Preparation of [11,12-³H]LXA₄. Tritiation of 11,12-acetylenic LXA₄methyl ester was carried out under a custom tritiation service(NET-259:92-2326) by New England Nuclear (Boston, Mass.). Briefly,11,12-acetylene methyl ester was characterized by UV absorbance andreverse-phase HPLC as in (Nicolaou, K. C. et al (1985) J. Am. Chem. Soc.107:7515) and exposed to tritium atmosphere in methylene chloride atroom temperature. This incubation was stirred in the presence of Lindlarcatalyst (1.0 mg from Fluka Chemicals) for ˜1 h. The resulting mixturewas stored in methanol and isolated using RP-HPLC. Tritiated productswere chromatographed as methyl esters utilizing a gradient HPLC systemequipped with a photodiode array rapid spectral detector (Serhan, C. N.,Methods in Enzymology: Arachidonate Related Lipid Mediators, in MurphyR. C., Fitzpatrick, F. (eds.), vol. 187. Orlando, Fla., Academic,(1990), p. 167). This mixture contained both [11,12-³H]LXA₄ and[11,12-³H]-11-trans-LXA₄ methyl esters (˜1:3 ratio) as determined bycoelution with synthetic standards. After RP-HPLC, fractions containing[11,12-³H]LXA₄ were collected, and extracted into ethyl acetate. Thefree acid was prepared by LiOH saponification (Fiore, S. et al. (1992)J. Biol. Chem. 267:16168). Material from these fractions, when injectedinto UV-electrochemical detection HPLC, gave greater than 90% ofradioactivity associated with a tetraene-containing product thatcoeluted with synthetic LXA₄. Materials that eluted with the retentiontime of authentic LXA₄ in two HPLC systems were taken for bindingexperiments. The specific activity calculated for [11,12-³H]LXA₄ was40.5 Ci/mmole.

Cell cultures and differentiation. HL-60 cells were seeded in RPMImedium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin,and 10% fetal calf serum (Hyclone, Logan, Utah), incubated at 37° C.with 5% CO₂ atmosphere in 250 ml flasks. Individual flasks containing˜50×106 cells/ml next received dimethyl sulfoxide (DMSO) (1.12% v/v, 120h) or retinoic acid (RA) (1 μM, 120 h) or phorbol myristate acetate(PMA) (20 nM, 48 h). Before performing binding assays, cells were washedtwice in phosphate buffered saline (PBS), Ca²⁺- and Mg²⁺-free,enumerated and suspended at 20×10⁶ cells/ml in Tris buffer (10 mM), pH7.4 (Fiore, S. et al. (1992) J. Biol. Chem. 267:16168). Nitro bluetetrazolium reduction was performed to monitor induction ofpolymorphonuclear phenotype as in (Imaizumi, M. and Breitman, T. R.(1986) Blood 67:1273), and cell adherence was determined for inductionof macrophage-like phenotype (Collins, S. J. (1987) Blood 70:1273).Human umbilical vein endothelial cells (HWEC) maintained in culturethird passage were obtained from Dr. M. Gimbrone (Brigham and Women'sHospital Department of Pathology).

PMN. platelet and RBC isolation from peripheral blood. Human PMN wereobtained by the modified Boyum method (Böyum, A. (1968) Scan. J. Clin.Lab. Invest. 21:77) from fresh heparinized blood after venipuncture ofhealthy normal volunteers. Suspensions in PBS were monitored for cellnumber and viability by their ability to exclude trypan blue bothexceeding 98%. Red blood cells were obtained from 10 ml of heparinizedblood after three centrifugations in PBS (2,500 rpm, 10 min at 21° C.).Blood drawn in acidic citrate dextrose (9:1, v/v) was used to isolateplatelets as previously described (Serhan, C. N. and Sheppard, K.-A.(1990) J. Clin. Invest. 85:772).

Lipand binding. ³H-LXA₄ and ³H-LTB₄ binding was performed essentially asin Fiore, S. et al. (1992) J. Biol. Chem. 267:16168. After suspendingcells in Tris buffer 10 mM (pH 7.4, Ca²⁺ 2.5 MM, Mg²⁺ 1.2 mM), aliquots(0.5 ml) were incubated (20 min at 4° C.) with ³H-ligands alone (0.3 nM)or in the presence of increasing concentrations of homoligands or othercompounds (3-300 nM). The incubations were rapidly centrifuged (60 sec,12,000 g) on silicon oil (d=1.028), and cell-associated radioactivitywas determined by liquid scintillation counting (Wallac 1409, Pharmacia,Piscataway, N.J.). Binding experiments with HUVEC cells were performedin 12 well plates with 3.5×10⁵ cells/well. After 10 min, wells werewashed twice with PBS and cell-associated label was recovered by addingglacial acetic acid (0.5 ml). Results obtained from these assays weresubmitted for further analysis using the Ligand program(Elsevier-Biosoft, Cambridge, UK).

PLD activity. Human PMN and HL-60 cells (50×10⁶ cells/ml) prepared asabove were incubated with ³H-myristic acid or ³H-palmitic acid (8μCi/50×106 cells) for 40-60 min at 37° C. in PBS. Cell uptake rangedbetween 60-80% of added label, 7.1±4.2% (n=10; mean±S.D.) and 32.6±10.3%(n=6; mean±S.D.) into total phospholipid classes of PMN and HL-60 cells,respectively. Incubations were carried out at 37° C. (10×10⁶ cells/mlPBS). Agonists were added in 50 μL PBS or with 1:10 (v/v) EtOH:PBS forphosphatidylethanol (PEt) formation as in Billah, M. M. et al. (1989) J.Biol. Chem. 264:17069. At indicated times, incubations were stopped byadding 3.5 ml of ice cold CHCl₃/MeOH (2/5, v/v) containing1-¹⁴C-arachidonic acid (5,000 cpm) used here as internal standard toquantitate extraction recoveries. Samples were extracted using amodified Bligh and Dyer extraction as in (Serhan, C. N. and Sheppard,K.-A. (1990) J. Clin. Invest. 85:772). Organic phases, concentrated in50 μL of CHCl₃/MeOH (8/2, v/v), were spotted onto linear K6D TLC platesdeveloped with the organic phase of ethyl acetate/isooctane/aceticacid/water (110/50/20/100, v/v/v/v) for 50 min (Billah, M. M. et al.(1989) J. Biol. Chem. 264:17069). In this system, phosphatidic acid (PA)gave an R_(f)=0.1±0.04 and PEt gave an R_(f)=0.56±0.04; n=38±S.D. thatwere clearly separated from other phospholipids (that remained at theorigin) or neutral lipids (R_(f)=0.75-0.90). Lipids were visualized withiodine vapor and identified by co-elution with authentic standards thatwere also spotted and chromatographed in each TLC plate. Regionscorresponding to PA, PEt, and internal standards were scraped andquantified by liquid scintillation counting. In addition to ³H-myristateor ³H-palmitate labeling, both [1-¹⁴C]arachidonate (0.25 μCi/30×10⁶ PMN)and ³²PγATP (20 μCi/50×10⁶ PMN) labeled cells were used to monitorLXA₄-stimulated formation of PA and PEt generation. In theseexperiments, PA was resolved using ethyl acetate/isooctane/acetic acid(45/15/10, v/v/v) as solvent system (Bocckino, S. B. et al. (1989) Anal.Biochem. 180:24) and gave an R_(f)=0.46±0.03 (n=15). All values reportedfor PEt formation in Tables 4 and 5 were calculated by subtracting thedpm obtained in the presence of agonist(s) alone minus those measured inthe presence of agonist(s) and 0.5% EtOH.

Impact of IAP and staurosnorine on LXA₄-induced PLD activity. PLDactivity assays, performed as described (vide supra), were preceded bycell exposure to either IAP or staurosporine. IAP treatment of PMN wasperformed as previously described (Nigam, S. et al. (1990) J. CellPhysiol. 143:512), and HL-60 cells were incubated in the presence orabsence of IAP (300 ng/ml) for 2 h at 37° C. (Kanaho, Y. et al. (1992)J. Biol. Chem. 267:23554). Aliquots (10⁷ cells/0.5 ml) were added to 0.4ml buffer. Next, incubated cells were exposed to either 100 μl ofvehicle (PBS, EtOH 0.04%) or LXA₄ (10⁻⁷ M and 10⁻⁹ M), in the presenceor absence of 0.5% EtOH.

Staurosporine (100 nM) was added to cell suspensions for 5 min at 37° C.before addition of LXA₄.

RESULTS

After preparation of synthetic [11,12-³H]LXA₄, its specific binding topromyelocytic cells (HL-60) was characterized and direct comparisonswith specific binding of [14,15-³H]LTB₄ were performed. When routinephenotypic markers were monitored, untreated HL-60 cells displayed a lowlevel of specific binding for both ³H-LXA₄ and ³H-LTB₄ ligands.Differentiation induced by exposure to either DMSO (1.12%) or retinoicacid (1 μM) for 5 days was accompanied with a three to fivefold increasein specific binding for both radioligands. PMA-treated cells (20 nM, 48h) displaying characteristics of macrophagic-like phenotype, i.e. NBTnegative cells that adhere to plastic (see Imaizumi, M. and Breitman, T.R. (1986) Blood 67:1273; Collins, S. J. (1987) Blood 70:1233), also ledto the appearance of specific binding with both ³H-LXA₄ and ³H-LTB₄.Equilibrium binding with ³H-LXA₄ at 4° C. was reached at 10 min thatremained virtually unchanged for the next 20 min.

To assess whether induction of specific binding for both ³H-ligandsrequired de novo synthesis, Actinomycin D (2 μg/ml) was added with PMAincubations. Actinomycin D blocked the PMA-induced increment in specificbinding for both labeled eicosanoids, suggesting inhibition of de novoprotein biosynthesis also blocked appearance of specific binding sites.The impact of protease and glycosidase treatments was assessed with bothdifferentiated HL-60 cells and human PMN for ³H-LXA₄ specific binding.Protease treatment reduced specific binding and provided additionalevidence in support of a protein component of LXA₄ specific bindingsites.

Results from isotherm binding assays (4° C., 10 min) with differentiatedHL-60 cells and [11,12-³H]LXA₄ (0.1-30 nM) showed that [11,12-³H]LXA₄specific binding sites gave a K_(d)=0.6±0.3 nM. A nonlinear portion ofthe Scatchard plot was observed for concentrations observed with LXA₄specific binding with human PMN (Fiore, S. et al. (1992) J. Biol. Chem.267:16168). Results obtained here with LTB₄ specific binding with HL-60cells, K_(d)=0.12 nM, are essentially in agreement with values recentlyreported by Harada (Xie, M. et al. (1991) J. Clin. Invest. 88:45),namely K_(d)=0.23 nM.

To further characterize the interactions of ³H-LXA₄ with its specificbinding sites, competition binding experiments were performed withdifferentiated HL-60 cells. LXA₄, LXB₄, LTB₄, LTC₄ and the leukotrienereceptor antagonists SKF 104353 (LTD₄ antagonist; Harada, Y. (1990)Hiroshima J. Med. Sci. 39:89) and ONO-4057 (LTB₄ antagonist; Gleason, J.G. et al. (1987) J. Med. Chem. 30:959) were assessed as potentialcompeting ligands. Neither LXB₄, LTB₄, nor trihydroxyheptanoic acid(methyl ester) (300 nM) were able to displace ³H-LXA₄ specific bindingwith differentiated HL-60 cells while LTC₄ caused ˜30% decrease ofspecific binding when added in 3 log molar excess. The finding that LXA₄(300 nM) was unable to compete with ³H-LTB₄ (0.3 nM) binding todifferentiated HL-60 cells suggests that LXA₄ and LTB₄ interact withseparate classes of specific binding sites. Leukotriene receptorantagonists SKF 104353 and ONO-4057 did not displace ³H-LXA₄ bindingwith differentiated HL-60 cells, but SKF 104353 and LTD₄ were effectivein competing the specific ³H-LXA₄ binding with HUVEC. HUVEC displayed aK_(d) of 11.0±2.6 nM and a B_(max) of 2.5×10⁻¹⁰ M for ³H-LXA₄, andvirtually identical values were calculated for LTD₄ competition. In thecase of ³H-LTB₄, HUVEC did not specifically bind LTB₄, but non-specificcell association was evident with this ³H-ligand (n=3; not shown).Specific association of ³H-LXA₄ was not evident among several other celltypes surveyed. Here, neither washed platelets, RBCs, the β-cell (Raji),nor T-cell (Jurkat) cultured cell lines displayed specific binding for³H-LXA₄. Taken together, these results indicate that LXA₄ interacts withunique binding sites in differentiated HL-60 cells that is not sensitiveto either leukotriene receptor antagonist (SKF 104353 or ONO-4057). InHUVEC, ³H-LXA₄ specific binding was sensitive to both LTD₄ and SKF104353 but not ONO-4057, suggesting that ³H-LXA₄ specific binding inthis cell type may reflect its interaction with putative LTD₄ receptors.

LXA₄ rapidly stimulates phosphatidic acid formation in human neutrophils(Nigam, S. et al. (1990) J. Cell Physiol. 143:512). To determine if³H-LXA₄ binding confers PLD activation, PEt and PA were monitored inboth PMN and HL-60 cells. Results indicated that LXA₄ stimulates PLDactivity in these cell types with similar temporal responses. PMNexposed to LXA₄ (10⁻¹⁰ M) rapidly generated the ethanol trapping productPEt within 60 s that declined to baseline levels by 5 min. In theabsence of added EtOH, PEt was not formed at statistically significantlevels. A biphasic concentration dependence was obtained for PEtformation in both PMN and differentiated HL-60 cells. An apparentmaximal response was noted with the concentration range of ˜10⁻⁹-10⁻¹⁰ MLXA₄, and a second peak of activity was observed at 10⁻⁷ M LXA₄. Below10⁻⁸ M, both the chemotactic peptide FMLP and LXA₄ gave results ofsimilar magnitude with FMLP appearing to be slightly more potent withPMN from some donors. To evaluate the potential contribution of otherbiosynthetic pathways, LXA₄-induced PA formation was also examined inboth ³²PγATP and [1-¹⁴C]-arachidonate-labeled PMN. ³²P-labeled PA wasevident in statistically significant levels only after 30 min ofexposure to LXA₄ (10⁻⁷ M). Similar results were obtained with¹⁴C-labeled PA formation derived from ¹⁴C arachidonate-labeledprecursors. These findings indicated that LXA₄ can also stimulate otherroutes of PA formation in PMN but only after 30 min of exposure.

Only differentiated HL-60 cells (expressing specific binding sites for³H-LXA₄) incubated with LXA₄ rapidly generated PEt that was evidentwithin 30 sec. Undifferentiated HL-60 cells incubated with LXA₄ (10⁻⁹ M)did not rapidly generate PEt. The concentration dependence with thesecells also gave a biphasic response with LXA₄ and gave an apparentmaximum at 10⁻⁹ M. To investigate possible signal transduction eventsinvolved in LXA₄-mediated PLD activation, PMN and HL-60 cells were nextexposed to either IAP or staurosporine. Results indicate that theLXA₄-mediated PLD activity evoked within the lower concentration range(10⁻⁹-10⁻¹⁰ M) was sensitive to IAP treatment in both cell types and,similarly, the PLD activity stimulated at higher concentrations of LXA₄(10⁻⁷ M) was inhibited by staurosporine. Thus, in both cellular systemsat concentrations below 10⁻⁸ M, LXA₄ rapidly interacts with specificbinding sites that trigger PLD activity and hence confers a functionalresponse, while within submicromolar concentrations of LXA₄, it maystimulate additional processes that can also lead to activation of PLD.

Example 4 Lipoxin Bioactivity Assays

Several of the preferred LX analogs (shown structurally as compounds 1through 8 above) were prepared by total synthesis as described inExample 1. Following preparation and isolation of these compounds viaHPLC, compounds were first assessed to determine whether they retainbiological activity using the neutrophil adhesion assay and epithelialcell transmigration assays (as described in Nash, S et al., (1987) J.Clin. Invest. 80:1104-1113; Nash, S et al., (1991) J. Clin. Invest. 87:1474-1477; Parkos, C. A. et al., (1991) J. Clin. Invest. 88:1605-1612;Parkos, C. A. et al. (1992) J. Cell. Biol. 117:757-764; Madara J. L. etal., (1992) J. Tiss. Cult. Meth. 14:209-216).

Compounds 1 through 8 (10⁻⁷-10⁻¹⁰M) were found to inhibit neutrophiladhesion to endothelial cells and their transmigration on epithelialcells. The acetylenic precursors (compound 1, 3, 5 and 7) were found tobe physically more stable than their tetraene counterparts. Compound 7,which did not have an alcohol group in the C15 position or othermodifications in the series, showed no biological activity in theassays. It would therefore appear that a substituent in the C15 positionof LX is necessary for the biological activity of at least LXA₄ analogs.15-methyl-LX A₄ (compound 2) also proved to inhibit polymorphonuclear(PMN) adhesion triggered by leukotriene B₄ (LTB₄) to human endothelialcells with an IC₅₀ of ˜1 nM. LX analogs 1 through 8 were found to blockmigration at potencies greater than or equal to synthetic LXA₄. Compound7 was found to be essentially inactive within the concentration rangefor inhibition induced by LXA₄ or other analogs. The results in theseneutrophil-containing bioassays indicate that LXA₄ analogs withmodifications in C15-C20 positions retain their biological action andcan inhibit PMN transmigration and adhesion events.

The “bio-half-life” of compounds 1-8 was assessed using phorbolester-treated human promyelocytic leukemia (HL-60) cells as described inExample 2. These cells converted more than 95% of LX A₄ within fiveminutes of its addition to the cell incubation. LXA₄ in this system wasrapidly transformed to 15-oxo-LXA₄. However, in the same assay,15-methyl-LXA₄ (compound 2) and cyclohexyl-LXA₄(compound 4) werequantitatively recovered in the incubation medium at times up to twohours. These results illustrate that modification in the carbon 20through the carbon 15 positions prevents the further metabolism of LXA₄by leukocytes.

In addition, the stability of the acetylenic methyl ester LXA₄(compound 1) was recovered essentially intact after 60 minutes ofincubation in whole blood (37° C.) ex vivo, as assessed after extractionand reverse phase HPLC. When taken together, these results indicate thatLX analogs retain biological action and are resistant to furthermetabolism in vitro.

Example 5 Effect of 15-epi-Lipoxins' on Cell Proliferation MATERIALS andMETHODS

Synthetic(5S,6R,15R)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoate:carboxymethylester (15-epi-LXA₄-methyl ester) was prepared by total organic synthesisand was a gift of Prof. N. A. Petasis (Department of Chemistry,University of Southern California). 15-epi-LXA₄ free acid was obtainedby saponification of 15-epi-LXA₄-methyl ester in tetrahydrofuran withLiOH (0.1 M) at 4° C. for 24 h. Synthetic eicosanoid reference sampleswere from Cascade Biochem Limited (Reading, Berkshire, England).Inhibitors of 5-LO (Rev 5901 isomer) and cytochrome P450(17-octadecaynoic acid, 17-ODYA) activities were from Biomol (PlymouthMeeting, Pa.). Radiolabeled ([³²P]) dCTP and ([³H]) arachidonic acid andmethyl-thymidine were from Dupont NEN (Boston, Mass.). Ionophore(A₂₃₁₈₇), ASA, 3,(4,5-dimethylthiazoyl-2-yl) 2,5 (diphenyl-tetrazoliumbromide) (MTT) and guanidinium isothyocyanate were purchased from SigmaChemical Co (St. Louis, Mo.). Recombinant human interleukin 1_(β)(IL-1_(β)) was obtained from R&D systems (Minneapolis, Minn.).Dulbecco's phosphate-buffered saline containing both CaCl₂ (0.6 mM) andMgCl₂ (1.0 mM) (pH 7.4) (DPBS²⁺), fetal bovine serum (FBS), penicillinand streptomycin were from Bio Whittaker (Walkersville, Md.). Hank'sbalanced salt solution (HBSS) and F-12K nutrient mixture were from GibcoLaboratories (Grand Island, N.Y.). High-pressure liquid chromatography(HPLC) grade solvents and cesium chloride were purchased from J T Baker(Phlllipsburg, N.J.), methyl formate was from Eastman Kodak Co(Rochester, N.Y.) and Sep-Pak C₁₈ cartridges were from Waters Associates(Milford, Mass.). Diazomethane was prepared fromN-methyl-N′-nitro-N-nitroguanidine purchased from Aldrich ChemicalCompany (Milwaukee, Wis.). N,0-bis (trimethylsilyl) trifluoroacetamide(BSTFA) was from Pierce (Rockford, Ill.). First strand cDNA synthesiskit and other molecular biology reagents were from Promega (Madison,Wis.). Oligonucleotide primers were purchased to Integrated DNATechnologies (Coralville, Iowa).

Cell isolation and culture:

Human type II epithelial A549 cells from human lung carcinoma and normalhuman skin fibroblast (breast) were obtained from the American TypeCulture Collection (Rockville, Md.). The A549 cell line originated froma human alveolar cell carcinoma and was a useful cell line because itwas easily assessable and could be maintained in culture withoutcontaminating tissue macrophages. (Lieber, M., et al. (1976) Acontinuous tumor-cell line from a human lung carcinoma with propertiesof type II alveolar epithelial cells. Int. J. Cancer Fibroblasts wereutilized within their limited window of viability and their passagenumbers were recorded (results are reported for cells from 3-5passages). Epithelial A549 cells were seeded into T-75 em² tissueculture flasks and maintained in F-12K medium supplemented with 10%heat-inactivated FBS, penicillin (50 U/mL) and streptomycin (50 (g/mL).Human PMN from healthy donors who had not taken ASA or other medicationsfor at least two weeks were obtained by Ficoll-Hypaque gradientcentrifugation and dextran sedimentation, (Boyum A. (1968) Isolation ofmononuclear cells and granulocytes from human blood. Isolation ofmononuclear cells by one centrifugation, and of granulocytes bycombining centrifugation and sedimentation at 1 g. Scand. J Clin. Lab.Invest. 21 (Suppl. 97): 77-89.), and suspended in DPBS⁺ at pH 7.4.Viability of A549 cells and PMN was determined by their ability toexclude trypan blue and were 95±2 and 97±1%, respectively. These valueswere not significantly altered during the reported incubations.

Incubation Conditions:

In incubations involving permeabilized A549 cells (prepared by a rapidfreeze-thaw cycle), IL-1_(β)-treated (1 ng/ml, 24 h) A549 cells (1.5×10⁶cells/ml) were pretreated for 20 min with either vehicle (0.1% EtOH),ASA (500 (M; used throughout), 5 (M 17-ODYA, an inhibitor of cytochromeP450, (Muerhoff A. S., et al. (1989) Prostaglandin and fatty acid ω and(ω-1)-oxidation in rabbit lung: acetylenic fatty acid mechanism-basedinactivators as specific inhibitors. J. Biol. Chem. 244: 749-756.), or 5mM Rev 5901 isomer, a 5-LO inhibitor, subjected to two cycles of rapidfreezing in a dry ice-acetone bath and thawing to room temperature (fullcycle <20 min), and incubated with arachidonic acid (20 (M) for 20 minat 37° C. in 4 ml of DPBS²⁺. In experiments involving radiolabeledarachidonic acid, incubations were initiated with the addition of[³H]-arachidonic acid (0.25 (Ci/ml) plus unlabeled arachidonic acid (20(M) for 20 min at 37° C.

For time-course experiments involving the generation of 15-HETE fromendogenous sources (see FIG. 2B), intact A549 cells were exposed toIL-1_(β) (1 ng/ml) for up to 48 h and then treated with vehicle(containing 0.1% EtOH) or ASA for 20 min followed by addition ofionophore A₂₃₁₈₇ (5 μM) in 4 ml of HBSS for 30 min at 37° C.

In coincubation experiments, confluent A549 cells were exposed toIL-1_(β) (1 ng/ml, 24 h), washed in HBSS and treated with vehicle aloneor ASA for 20 min and arachidonic acid (20 (M) for 60 seconds at 37° C.Coincubations were performed by adding PMN to A549 cell monolayersfollowed by costimulation with ionophore A₂₃₁₈₇ (5 μM) in 4 ml of HBSSfor 30 min at 37° C.

Analysis of eicosanoids:

Incubations were stopped with 2 volumes cold MeOH containingprostaglandin B₂ (200 ng) and products were extracted using Sep-Pak C₁₈cartridges. Materials which eluted in the methyl formate fractions wereconcentrated under a stream of N₂ and scanned for ultraviolet-absorbingmaterial (in methanol) with a model 8452 spectrophotometer (HewlettPackard Co, Palo Alto, Calif.) prior to injection into a reversed phase(RP)-HPLC system. This system consisted of a dual pump gradient (LKB,Bromma, Sweden), a diode array detector (Hewlett-Packard 1040M seriesII) and a HPLC^(3D) ChemStation software. The collected UV data wererecalled at 300 nm to monitor conjugated tetraenes, at 270 nm fortrienes and 234 for monoHETEs. All UV spectra were acquired using step=4nm, Bw=10 nm, and range=220-360 nm with a sampling interval of 0.96 s.

The monohydroxy eicosanoids (i.e. 5-, 12- and 15-HETE) from A549 cellswere analyzed using a Ultrasphere-ODS column (5 μm, 4.6 mm×25 cm)(Beckman Instruments, Fullerton, Calif.) was eluted with MeOH/H₂O/aceticacid (65:35:0.01; v/v/v) as phase one (t₀-20 min), and a linear gradientwith MeOH/acetic acid (99.9:0.1, v/v) as phase two (20-45 min) at a flowrate of 1.0 ml/min. The R- and S-enantiomers of 15-HETE were resolvedand identified using a chiral HPLC system similar to that reported byHawkins et al. (1988). (Hawkins et al. (1988) Resolution of enantiomersof hydroxyeicosatetraenoate derivatives by chiral phase high-pressureliquid chromatography. Anal. Biochem. 173: 456462.) Briefly, afterRP-HPLC material eluting beneath 15-HETE peak was extracted withchloroform and converted into methyl ester by ethereal diazomethanetreatment, chiral analysis was performed with a Bakerbond DNBPG(covalent) chiral column (5 μm, 4.6 mm×25 cm) (J T Baker, Phillipsburg,N.J.) eluted with n-hexane/2-propanol (100:0.4; v/v) at a flow rate of0.8 ml/min. When indicated, generation of 15-HETE from endogenoussources was monitored by radioimmunoassay (RIA). The antibody was raisedagainst 15S-HETE with 0.1% crossreactivity at 50% B/B₀ for 5-HETE(PerSeptive Diagnostics, Cambridge, Mass.).

For analysis of LXs (including LXs and 15-epi-LXs) from A549 cell-PMN,coincubations were carried out using either a Waters (Bondapak C₁₈(3.9×300 nm) column eluted with an isocratic mobile phaseMeOH/H₂O/acetic acid (60:40:0.01; v/v/v) with a flow rate of 0.6 ml/minor an Altex Ultrasphere ODS column (5 μm, 10 mm×25 cm) eluted withMeOH/H₂O/acetic acid (65:35:0.01; v/v/v) at a flow of 3 ml/min.Peptidoleukotrienes (LTC₄ and LTD₄) eluted in the MeOH fractions fromSep-Pak cartridge extractions were resolved with a BeckmanUltrasphere-ODS column eluted with MeOH/H₂O/acetic acid (65:35:0.01;v/v/v), pH 5.7, at 1 ml/min. Incubations of PMN with 15R-HETE werestopped with MeOH and methyl formate fractions of the Sep-Pak C₁₈extracted products were injected into an Altex Ultrasphere ODS column (5μm, 10 mm×25 cm) eluted with MeOH/H₂O/acetic acid (65:35:0.01; v/v/v)using a flow of 3 ml/min. The material beneath peaks absorbing at 300 nmwere individually collected after RP-HPLC and analyzed by gaschromatography-mass spectrometry (GC-MS) employing a Hewlett-Packard5890 GC series II equipped with a 5971A mass-selective quadrapoledetector as in Claria, J. and Serhan, C. N. (1995) Aspirin triggerspreviously undescribed bioactive eicosanoids by human endothelialcell-leukocyte interaction. Proc. Natl Acad. Sci. USA 92:9475-9479,herein expressly incorporated by reference.

Reverse Transcription (RT) and PCR:

Total RNA was obtained from A549 cells by the guanidiniumisothiocyanate-cesium chloride method and cDNA was produced by RT.Oligonucleotide primers were constructed from published sequences ofPGHS-1 and PGHS-2 ((5′-TGC CCA GCT CCT GGC CCG CCG CTT-3′ (sense),5′-GTG CAT CAA CAC AGG CGC CTC TTC-3′ (antisense)), and (5′-TTC AAA TGAGAT TGT GGG AAA ATT GCT-3′ (sense) and 5′-AGA TCA TCT CTG CCT GAG TATCTT-3′ (antisense)), respectively), 15-LO (5′-ATG GGT CTC TAC CGC ATCCGC GTG TCC ACT-3′ (sense) and 5′-CAC CCA GCG GTA ACA AGG GAA CCT GACCTC-3′ (antisense)), 12-LO, (Funk, C. D. and FitzGerald, G. A. (1991)Eicosanoid forming enzyme mRNA in human tissues. J. Biol. Chem. 266:12508-12513), (5′-AGT TCC TCA ATG GTG CCA AC-3′ (sense) and 5′-ACA GTGTTG GGG TTG GAG AG-3′ (antisense)) and 5-LO (5′-GAA GAC CTG ATG TTT GGCTACC-3′ (sense) and 5′-AGG GTT CTC ATC TCC CGG-3′ (antisense)).Amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)primers was performed with 5′-CCA CCC ATG GCA AAT TCC ATG GCA-3′ (sense)and 5′-TCT AGA CGG CAG GTC AGG TCC ACC-3′ (antisense). PGHS-1, PGHS-2and GAPDH samples were amplified for 25 cycles of denaturation at 94° C.for 1 min, annealing at 58° C. for 2 min and extension at 72° C. for 3min. 5-, 12- and 15-LO were amplified at 94° C. (1 min), 55° C. (2 min)and 72° C. (2.5 min) for 35 cycles. PCR products were analyzed byelectrophoresis in 2% agarose gel and their identity was monitored byrestriction enzyme analysis. For detection of specific PCR-amplifiedtargets, 0.5 (Ci of [³²P]dCTP (3000 Ci/mmol) was added to the PCRmixture and the products were quantified by phosphorimager usingImage-Quant programming (Molecular Dynamics, San Lorenzo, Calif.).

Cell Proliferation:

A microculture 3,(4,5-dimethylthiazoyl-2-yl) 2,5 (diphenyltetrazoliumbromide) (MTT) assay, (Marshall, N.J., et al (1995) A criticalassessment of the use of microculture tetrazolium assays to measure cellgrowth and function. Growth Regul. 5: 69-84.), was used to examine theactions of LXs and other eicosanoids on cell proliferation. A549 cellsand fibroblasts from exponential-phase maintenance cultures were countedand dispensed within replicate 96-well culture plates in 100 μl volumesof medium (˜2000 cells/well). Following 24 hours at 37° C., the culturemedium was removed, and fresh medium (100 μl) containing either thecompounds (5-1000 nM) or vehicle (medium plus 0.15% ethanol) was addedto 4 replicates for each condition studied and the culture plates werethen incubated for up to 96 hours at 37° C. in a 5% CO₂ atmosphere. Atthe end of this period, 25 μl of freshly prepared MTT in HBSS (5 mg/ml)was added to the wells and the plates were incubated for 4 hours at 37°C. Dye solution was aspirated, wells were washed once with HBSS and dyetaken up by the cells was extracted in 100 μl of isopropyl alcohol:1 NHCl (96:4, v/v) and quantitated at 570 nm using a microplate reader(Molecular Devices, Menlo Park, Calif.). In some experiments, cells weregrown in 12-well culture plates, treated as above and enumerated using aNeubauer hemocytometer. Viability was assessed routinely using trypanblue exclusion assay. For the A549 cells and fibroblasts, a linearrelation was established for the MTT values and cell number within therange of the experiments shown (r=0.995, P<0.005). A549 cells grown for72 hours in the presence of the compounds (5-1000 nM) or vehicle (0.15%EtOH) were lysed with 0.25 N NaOH and the cellular protein content wasdetermined by applying the Bio-R_(a)d (Richmond, Calif.) microassaymethod using bovine serum albumin as standard. The mean cellular proteincontent in resting A549 cells was 46.6±1.5 pg/cell.

Thymidine incorporation and DNA synthesis:

A549 cells (˜2×10⁴ cells/ml) were seeded in 96-well plates, allowed tosettle for 24 hours and grown for an additional 72 hours in the presenceof compounds (5-1000 nM) or vehicle (medium plus 0.15% ethanol).Twenty-four hours before the assay, 2 (Ci/ml of methyl-[³H]thymidine(specific activity 6.7 Ci/mmol) were added to each well. (See Cybulskyet al. (1992) Eicosanoids enhance epidermal growth factor receptoractivation and proliferation in glomerular epithelial cells. Am. J.Physiol. 262 (Renal Fluid Electrolyte Physiol.31): F639-F646.) Afterpulse-labeling, each well was washed four times with cold DPBS²⁺, andthe cells were lysed with NaOH (0.25 N) and radioactivity measured.

The Student's t-test was used for statistical analysis and differenceswere considered significant at a P value (0.05).

RESULTS

Eicosanoids are formed by initial oxygenation of arachidonic acid byPGHS or LO enzymatic pathways. (Samuelsson B., et al. (1987)Leukotrienes and Lipoxins: structures, biosynthesis, and biologicaleffects. Science 237: 1171-1176.) To assess which of theeicosanoid-generating enzymes are present and/or regulated by cytokinesin A459 cells, mRNA levels of PGHS-1 and -2 and 5-, 12- and 15-LO fromA549 cells grown in the presence or absence of IL-1_(β) were monitoredby RT-PCR followed by phosphorimager analysis. As illustrated in FIG.1A, mRNA levels for PGHS-2 were significantly increased (˜twofold) afterstimulation of A549 cells with IL-1_(β). In contrast, mRNA levels forPGHS-1 and 5-LO were not significantly altered after exposure of thecells to cytokine (FIG. 1A). A549 cells failed to show either 15- or12-LO expression before or after cytokine induction (FIG. 1A). Theabsence of 15-LO mRNA in A549 cells, was further confirmed by performingthe RT-PCR in parallel with human lung tissue and peripheral bloodmonocyte RNA, which are known positive and negative sources, (cf. FunkC. D. and FitzGerald G. A. (1991) Eicosanoid forming enzyme mRNA inhuman tissues. J. Biol. Chem. 266: 12508-12513), of 15-LO mRNA,respectively (see FIG. 1A, inset).

To characterize the profile of monohydroxy products produced by airwayepithelial cells, IL-₁ _(β)-stimulated A549 cells (1.5×10⁶ cells/ml)were permeabilized and incubated with arachidonic acid, and the productsformed were extracted and analyzed by RP-HPLC. The chromatographicprofile revealed the presence of a major product with strong UVabsorbance at 234 nm which coeluted with synthetic 15-HETE. Also, when[³H]-achidonic acid was added to IL-1_(β)-treated A549 cells,radiolabeled material was recovered beneath the peak coeluting with15-HETE (FIG. 1B). In these experiments, the formation of either 5- or12-HETE was not consistently observed. ASA treatment of A549 cells ledto a marked increase in the formation of 15-HETE, while incubation ofpermeabilized A549 cells with 17-ODYA, a reported inhibitor of P450eicosanoid metabolism, (Muerhoff, A. S. et al. (1989) Prostaglandin andfatty acid ω and (ω-1)-oxidation in rabbit lung: acetylenic fatty acidmechanism-based inactivators as specific inhibitors, J. Biol. Chem.244:749-756), resulted in 50% reduction in 15-HETE (FIG. 2A). 17-ODYA isa potent inhibitor of P450 eicosanoid metabolism and does notselectively inhibit either cyclooxygenase or LO activity. (See Muerhoffet al. and supplier's supporting materials.) The 5-LO inhibitor(Rev-5901 isomer) did not alter the amount of 15-HETE produced by A549cells in a statistically significant fashion. Heat-denatured A549 cellsreduced the quantities of 15-HETE by ˜90%, suggesting an enzymaticcomponent in its formation. Production of 15-HETE from endogenoussources of arachidonate was also obtained from intact A549 cells(25.0±10.0 ng/10⁷ cells) treated with IL-1 (1 ng/ml) for 24 hours. Takentogether these results indicate that 15-HETE is the main monohydroxyproduct generated by A549 cells and suggest that acetylated PGHS-2 andcytochrome P450 each contributes to its biosynthesis.

To investigate the time-course for generation of 15-HETE from endogenoussources of arachidonate, intact A549 cells were exposed to IL-1_(β) (1ng/ml) for up to 48 h and the amount of immunoreactive 15-HETE presentin the cell supernatant was monitored by means of a specific RIA. Inresting conditions, A549 cells produced significant levels ofimmunoreactive 15-HETE (19.1±10.5 ng/10⁷ cells, n=3, d=2). These valueswere unchanged by addition of ionophore A₂₃₁₈₇ (5 (M) in the absence ofIL-1_(β) (FIG. 2B). Also, in the absence of ionophore stimulation,addition of IL-1_(β) to A549 cells for up to 48 h did not result in anaugmented generation of 15-HETE (FIG. 2B). In sharp contrast, additionof IL-1_(β) plus A₂₃₁₈₇ stimulation of A549 cells led to a markedincrease in the production of 15-HETE (FIG. 2B). The maximal levels werefound at 24 h of exposure to the cytokine with the levels of 15-HETEdeclining thereafter.

Because the stereochemistry of the alcohol in 15-HETE produced by A549cells was of interest, the relative amounts of individual R and Senantiomers of 15-HETE generated by IL-1_(β)-treated A549 cells wereexamined using a chiral phase HPLC analysis. (See Methods.) PGHS-2 aswell as cytochrome P450 are enzymes other than 15-LO that can eachconvert arachidonic acid to 15-HETE. ASA-acetylated PGHS-2-derived15-HETE carries its carbon (C)-15 alcohol group mainly in the Rconfiguration. (Holtzman, M. J., et al., (1992) Identification of apharmacologically distinct prostaglandin H synthase in culturedepithelial cells, J. Biol. Chem. 267:21438-21445). Here, 15-HETEproduced by IL-1_(β)-primed A549 cells was converted to its methyl esterand subjected to SP-HPLC chiral analysis. The 15-HETE from activatedA549 cells was 65% in the R and 35% in the S configuration (FIG. 3).Pretreatment of A549 cells with ASA (20 min, 37° C.) resulted in a3-fold increase in the amounts of 15R-HETE whereas formation of 15S-HETEremained unaltered (FIG. 3). In the presence of ASA, 15R-HETE accountedfor 85% of the total amount of 15-HETE produced by A549 cells. Theseresults indicate that the majority of 15-HETE generated byIL-1_(β)-primed A549 cells in presence of ASA was in the Rconfiguration.

Transcellular eicosanoid biosynthesis is an important means ofamplifying lipid mediators as well as generating new mediators. (Marcus,A. J. (1995) Aspirin as prophylaxis against colorectal cancer, N. Engl.J. Med. 333:656-658.) Costimulation of human endothelial cells and PMNafter ASA treatment results in the formation of a new class of bioactiveeicosanoids. (Claria, J. and Serhan, C. N. (1995) Aspirin triggerspreviously undescribed bioactive eicosanoids by human endothelialcell-leukocyte interactions, Proc. Natl. Acad. Sci. USA 92:9475-9479.)These novel eicosanoids were identified as 15-epi-LXs and theirbiosynthesis involve leukocyte transformation of ASA-triggeredendothelial-derived 15R-HETE. In view of these results, it is possiblethat formation of new eicosanoids by transcellular biosynthesis alsooccurs during epithelial cell-PMN interactions. To test this hypothesis,confluent A549 cells were exposed to IL-1_(β) (1 ng/ml, 24 h), treatedwith ASA and costimulated with PMN. FIG. 4A shows a representative HPLCprofile of material obtained from stimulated cells exposed to ASA, whichrevealed the presence of four major products with strong UV absorbancewhen plotted at 300 mm. On-line spectral analysis of these productsshowed that they each displayed a triplet of absorbing bandscharacteristic of conjugated tetraene-containing chromophores indicativeof the LX basic structure (maxima at 301 nm and shoulders at 288 and316±2 mm) (FIGS. 4B and 4C).

LXA₄ and 15-epi-LXA₄ were identified in the chromatographic profiles onthe basis of coelution with synthetic standards and the presence of thecharacteristic chromophore. In these coincubations, 15-epi-LXA₄accounted for ˜88% of the total amount of LXA₄ detected which is inagreement with the observation that the majority of 15-HETE generated byIL-1_(β)-primed A549 cells exposed to ASA is predominantly in the Rconfiguration. (Cf. FIGS. 3 and 4A, 4B and 4C). In this RP-HPLC system,15-epi-11-trans-LXA₄ and LXB₄ coeluted, as did 11-trans-LXA₄ and15-epi-LXB₄ (not shown). These LX isomers were not further resolved byHPLC and are denoted in the profile beneath peaks labeled as peaks A andB, respectively (FIG. 4A). The compounds beneath peaks A and B didresolve as OTMS, methyl ester derivatives in GC-MS (vide infra). Theproducts were present in ˜8:2 ratio in favor of their 15R epimers (n=3).Compound C (FIG. 4A) did not coelute with any of the previouslyidentified LXs and was also present in the RP-HPLC profile fromactivated PMN incubated with 15R-HETE (data not shown, n=5). Materialeluting beneath Compound C matched the physical properties of CompoundIII that was recently isolated from endothelial cell-PMN interactions.(Cf. Clària, J. and Serhan, C. N. (1995) Aspirin triggers previouslyundescribed bioactive eicosanoids by human endothelial cell-leukocyteinteractions, Proc. Natl. Acad. Sci. USA 92:9475-9479.) Although thecomplete stereochemistry of compound C remains to be determined, the UVspectral data and chromatographic mobilities suggest that it may be the15-epimer form of 7-cis-11-trans-LXA₄. (Nicolaou, K. C., et al. (1989)Identification of a novel 7-cis-11-translipoxin A₄, generated by humanneutrophils: total synthesis, spasmogenic activities and comparison withother geometric isomers of LXs A₄ and B4, Biochim. Biophys. Acta1003:44-53.) Thus, the LXs generated during epithelial (A549 cell)-PMNcostimulation after ASA treatment were predominantly 15-epi-LX (FIG.4A).

In addition to the ability to produce LXs, coincubations of activatedPMN with A549 cells also generate significant amounts (8 times more thantetraene-containing LXs) of peptidoleukotrienes (pLTs; LTC₄, and LTD₄)in the absence of ASA (FIGS. 5A and 5B). The amounts of both LXs andpLTs produced in these coincubations were dependent upon individual cellratios (FIGS. 5A and 5B, insets). Exposure of airway epithelial A549cells to ASA (20 min) before addition of PMN led to an increase in theformation of LXs and a decrease in pLTs (FIGS. 5A and 5B). Neither PMNnor A549 cells incubated separately, in the absence or presence of ASA,generate detectable levels of LXs or pLTs (FIGS. 5A and 5B and data notshown). Taken together, these results indicate that, during A549cell-PMN interactions, both LXs and pLTs originate from transcellularroutes.

LXs are vasodilators and potent regulators of leukocyte responses, suchas inhibition of chemotaxis, adhesion to endothelial cells andtransmigration across epithelium. (See Serhan, C. N. (1994) Lipoxinbiosynthesis and its impact in inflammatory and vascular events.biochim. Biophys. Acta 1212:1-25.) In contrast, pLTs possess bothvasoconstrictor and proinflammatory actions as well as stimulate thegrowth of several cell types including fibroblasts, smooth-muscle andglomerular epithelial cells. (Baud, L., et al. (1985) Leukotriene C₄binds to human glomerular epithelial cells and promotes theirproliferation in vitro. J. Clin. Invest. 76:374-377.) LXs reverse thevasoconstrictor action of LTD₄ in rat renal hemodynamics and blockLTC₄-stimulated hematopoiesis. (Serhan, C. N. (1994) Lipoxinbiosynthesis and its impact in inflammatory and vascular events.Biochim. Biophys. Acta 1212:1-25.) Because ASA enhances 15-epi-LXformation and inhibits pLT biosynthesis (FIGS. 5A and 5B), theseeicosanoids may play counterregulatory actions on cell proliferation andcontribute to ASA's protective mechanisms in human cancer. To this end,the effect of these LO products on epithelial cell proliferation (FIGS.6A and 6B) was tested and their actions was compared to that ofdexamethasone, a well-established inhibitor, employing a solublemicroculture tetrazolium (MTT) assay. (Alley, M. C., et al. (1988)Feasibility of drug screening with panels of human tumor cell linesusing a microculture tetrazolium assay. Cancer Res. 48:589-601.) Theseexperiments were performed with synthetic LXA₄ and LXB₄, which wereavailable in sufficient quantities for bioassay, rather than the15-epi-LX, which are the major LX produced by these cells. As shown inFIGS. 6A and 6B, LXA₄ and LXB₄ inhibited A549 cell proliferation in atime- (A) and dose-dependent (B) fashion. LXA₄ and LXB₄ as well asdexamethasone [1 μM] inhibited A549 cell proliferation after 72 and 96hours of treatment (FIG. 6A). After 72 hours, LXA₄ shared theanti-proliferative properties (FIG. 6A) observed for dexamethasone withthese cells. (Cf. Croxtall, J. D., and Flower R. J. (1992) Lipocortin 1mediates dexamethasone-induced growth arrest of the A549 lungadenocarcinoma cell line. Proc. Natl. Sci USA 89:3571-3575.) The halfmaximum inhibition (IC₅₀) for LXA₄ was ˜80 nM compared to that ofdexamethasone, which was ˜7 nM. LXB₄ at concentrations of 0.5 and 1 μMwas (3 times more active than either LXA₄ or dexamethasone (FIG. 6B). Ofinterest, both LXA₄ and LXB₄ showed essentially equal potency forblocking A549 cell growth when each was added repeatedly (i.e., 24-hourintervals) to the cells for 3 consecutive days (data not shown, n=3,d=4), suggesting that LX may be inactivated by these epithelial cells.Results from additional experiments employing direct cell enumeration(FIG. 7B) and measurement of total cellular protein content (data notshown, n=3, d=4) paralleled those obtained with MTT assay, thusconfirming the anti-proliferative actions of LXs in A549 cells.Furthermore, a blockage of DNA synthesis, as determined by ³H-thymidineincorporation, occurred when A549 cells were exposed for 72 hours toconcentrations of 50 nM or higher of LXB₄ or dexamethasone (FIG. 7A).After incubation of A549 cells with the compounds, the viability of thecells, as determined by trypan blue exclusion assay, was found to be˜98%, indicating that these compounds were not cytotoxic within therange of concentrations used in these experiments.

The 15-hydroxy epimeric forms of LXA₄ and LXB₄ (15-epi-LXA₄ and15-epi-LXB₄, respectively), which were the dominant forms of LX isolatedfrom these cells, proved to also be potent inhibitors of epithelial cellproliferation, as shown in the following Table 1.

TABLE 1 LX and 15-epi-LX actions on cell proliferation Compound [10⁻⁷ %inhibition P value* Vehicle  1.6 ± 10.3 NS LXA₄ 16.3 ± 5.2 NS15-epi-LXA₄ 20.1 ± 1.9 <0.025 LXB₄ 30.0 ± 8.5 <0.005 15-epi-LXB₄ 79.3 ±0.3 <0.001^(&) Dexamethasone 42.1 ± 5.4 <0.005

Cells (2000 A549 cells/well) were grown in 96-well plates and exposed tovehicle (0.15% vol/vol in EtOH/F-12K media) or equimolar concentrations(10⁻⁷ M) of LXA₄, 15-epi-LXA₄, LXB₄, 15-epi-LXB₄, or dexamethasone for72 hours at 37 ° C. Values represent ±SEM from 3 to 7 experimentsperformed in quadruplicate and are expressed as percent inhibition ofcell growth. *P values denot statical differences as compared to cellsalone. & P<0.001 for 15-epi-LXB₄.

At equimolar levels (100 nM), 15-epi-LXA₄ inhibited A549 cell growth toa similar extent as LXA₄. On the other hand, 15-epi-LXB₄ isolated fromconversion of 15R-HETE by activated PMN and added back to the A549 cellsgave a more potent anti-proliferative activity than LXB₄ (˜80% vs. ˜34%inhibition proliferation, P<0.001; Table 1). This compound wascharacterized by UV, HPLC and GC-MS (C value: 23.3). Diagnostic ions forthe OTMS methyl ester were m/z 173 (base peak), 203, 289, 379 and lessprominent ions at 482 (M⁺-100) [It was not possible to obtain itsmolecular ion because of its low abundance]. Thus, the predominantmaterial beneath the peak labeled B in FIG. 4B was consistent with thatof 15-epi-LXB₄, which gave a shorter C value and separated from LXB₄ asOTMS, methyl ester derivative in GC-MS analysis. The mass spectra of15-epi-LXB₄ and LXB₄ were essentially identical (not shown) but their Cvalues were distinct. Material eluting beneath the peak denoted as C(isolated from activated PMN incubated with 15R-HETE) also showed a mildinhibitory action on epithelial cell growth (18±2% inhibitionproliferation, n=3, d=4). In sharp contrast, 15-epi-trans-LXA₄,11-trans-LXB₄, 8,9-acetylenic-LXB₄, peptidoleukotrienes (LTC₄ and LTD₄)and the LX precursors (15S- and 15R-HETE) each tested at 10⁻⁶-10⁻⁹ Mwere not able to significantly inhibit A549 cell proliferation (data notshown, n=3-5, d=4). These results indicate that LX and 15-epi-LX gave astereoselective action in blocking cell proliferation in A549 cells.LXA₄ and LXB₄ was tested with human skin fibroblasts to determine ifthey were antiproliferative for this cell type. At 100 nM, both LXA₄ andLXB₄ inhibited proliferation of fibroblasts. LXB₄ gave 38.0 (7.5%inhibition and LXA₄ 10.7 (1.8% compared to dexamethasone (29.7 (0.4%) aspositive control (n=3).

The presence of an active cytochrome P450 enzyme system in human airwayA549 cells, (Vogel, et al. (1994) Transforming growth factor-β1 inhibitsTCDD-induced cytochrome P450IA1 expressions in human lung cancer A549cells. Arch. Toxicol. 68: 303-307.), together with the results thatinhibition of P450 as well as heat denaturing blocks 15-HETE generationin these cells (FIG. 2A) suggests that this enzyme system in epithelialcells also contributes to 15-HETE biosynthesis and the generation of15-epi-lipoxin by transcellular routes (FIG. 8). Taken together, theseobservations (FIGS. 1-4) establish the existence of two separateenzymatic pathways (i.e. ASA-acetylated PGHS-2 and cytochrome P450),which can initiate the formation of 15-epi-lipoxin during airwayepithelial cell-PMN interactions (FIG. 8). Also, it should be notedthat, in view of ASA's ability to induce P450 enzymes, Pankow, D. et al.(1994) Acetylsalicylic acid-inducer of cytochrome P450 2E1? Arch.Toxicol. 68: 261-265, it is possible that these two independent routesmay act in concert to generate 15-epi-lipoxin.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 12(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 24 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:TGCCCAGCTC CTGGCCCGCC GCTT           #                  #                24 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 24 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:GTGCATCAAC ACAGGCGCCT CTTC           #                  #                24 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:TTCAAATGAG ATTGTGGGAA AATTGCT           #                  #             27 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 24 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4:AGATCATCTC TGCCTGAGTA TCTT           #                  #                24 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5:ATGGGTCTCT ACCGCATCCG CGTGTCCACT          #                  #           30 (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6:CACCCAGCGG TAACAAGGGA ACCTGACCTC          #                  #           30 (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7:AGTTCCTCAA TGGTGCCAAC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8:ACAGTGTTGG GGTTGGAGAG             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 22 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #9:GAAGACCTGA TGTTTGGCTA CC            #                  #                 22 (2) INFORMATION FOR SEQ ID NO: 10:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 18 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #10:AGGGTTCTCA TCTCCCGG              #                   #                  #  18 (2) INFORMATION FOR SEQ ID NO: 11:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 24 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #11:CCACCCATGG CAAATTCCAT GGCA           #                  #                24 (2) INFORMATION FOR SEQ ID NO: 12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 24 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #12:TCTAGACGGC AGGTCAGGTC CACC           #                  #                24

I claim:
 1. A compound of the structural formula:

wherein X is OR₁; wherein R₁ is (i) a hydrogen atom; or (ii) an alkyl of1 to 8 carbon atoms, inclusive, which may be straight chain or branched;wherein Q₁ is (C═O); wherein Q₃ is O; wherein R₂, R₃, R₄ are eachhydrogen atoms; wherein Y₁ or Y₂ is —OH and the other is H; wherein R₅is —R_(a)Q_(a)R_(b) wherein Q_(a)=—O—; wherein R_(a) is an alkylene of 0to 6 carbon atoms, inclusive, which may be straight chain or branched;wherein R_(b) is either alkyl of 0 to 8 carbon atoms, inclusive, whichmay be straight chain or branched or a substituted phenyl; wherein R₆ isa hydrogen atom; and pharmaceutically acceptable salts thereof.
 2. Thelipoxin compound of claim 1 wherein R₅ is —R_(a)Q_(a)R_(b) whereinQ_(a)=—O—; wherein R_(a) is alkylene of 0 to 6 carbon atoms, inclusive,which may be straight chain or branched; wherein R_(b) is either alkylof 0 to 8 carbon atoms, inclusive, which may be straight chain orbranched or substituted phenyl.
 3. The lipoxin compound of claim 2wherein the substituted phenyl is substituted by one or moresubstituents.
 4. The lipoxin compound of claim 3, wherein the phenylgroup is substituted with five substitutents.
 5. The lipoxin compound ofclaim 4, wherein each substituent is a halogen atom.
 6. The lipoxincompound of claim 5, wherein each halogen atom is a fluorine atom. 7.The lipoxin compound of claim 3, wherein the phenyl group is substitutedwith four substituents.
 8. The lipoxin compound of claim 7, wherein eachsubstituent is a halogen atom.
 9. The lipoxin compound of claim 8,wherein each halogen atom is a fluorine atom.
 10. The lipoxin compoundof claim 3, wherein the phenyl group is substituted with threesubstituents.
 11. The lipoxin compound of claim 10, wherein eachsubstituent is a halogen atom.
 12. The lipoxin compound of claim 11,wherein each halogen atom is a fluorine atom.
 13. The lipoxin compoundof claim 3, wherein the phenyl group is substituted with twosubstituents.
 14. The lipoxin compound of claim 13, wherein eachsubstituent is a halogen atom.
 15. The lipoxin compound of claim 14,wherein each halogen atom is a fluorine atom.
 16. The lipoxin analog ofclaim 3, wherein the phenyl group is substituted with one substituent.17. The lipoxin analog of claim 16, wherein the substituent is a halogenatom.
 18. The lipoxin analog of claim 17, wherein the halogen atom is afluorine atom.